CN105612174A

CN105612174A - Disulfide cyclic polypeptides for the treatment of heart failure

Info

Publication number: CN105612174A
Application number: CN201480052701.8A
Authority: CN
Inventors: F·泽克里; P·格罗舍; 八十岛华杨; H·赵; J·袁
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2013-07-25
Filing date: 2014-07-21
Publication date: 2016-05-25
Also published as: US9683018B2; EP3024847A1; US20160145309A1; JP2016531110A; JP6505692B2; WO2015013167A1

Abstract

The invention provides a synthetic polypeptide of Formula I': X1-R-P-R-X5-X6-X7-K-X9-P-X11-X12-X13 or an amide, an ester, a salt or a bioconjugate thereof, wherein X1, X5, X6, X7, X9 and X11 to X13 are defined herein. The polypeptides and bioconjugates are agonist of the APJ receptor. The invention also relates to a method for manufacturing the polypeptides or bioconjugates of the invention, and its therapeutic uses such as treatment or prevention of acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral arterial disease, cerebrovascular accidents, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn) and preeclampsia. The present invention further provides a combination of pharmacologically active agents and a pharmaceutical composition.

Description

Be used for the treatment of disulphide ring type polypeptide in heart failure

Technical field

The peptide that the present invention relates to comprise modification and the new compositions of peptide sequence, described peptide and peptide sequence are designed toTreat it and grant individual angiocardiopathy, and show better repellence and the equivalence to degraded than its wild type counterpartsOr higher biologically active. The invention still further relates to the described composition of preparation and control as pharmaceutically active agents with described compositionTreat the method for angiocardiopathy.

Background of invention

In the Western countries, the over-65s incidence of disease in heart failure of being grown up is approximately 1/100. Most commonly encountered diseases reason is cardiac muscle receiptsThe chronic shortage of contracting power and therefore cardiac output (effective blood volume that, arbitrary ventricle is discharged in time). Suffers from the chronic heartThe patient of force failure in the time that myocardial contractive power further declines, can have acute decompensation outbreak (, heart cannot maintain footEnough blood circulations). Only the U.S. just have an appointment every year 500000 people because of " acute decompensation DHF " (ADHF) be in hospital.

At present comprise diuretics, vasodilator for the therapy of ADHF and directly increase the cardiotonic of myocardial contractive power(inotropes). Current intravenous cardiotonic (dobutamine (dobutamine), dopamine (dopamine), milrinone(milrinone), Levosimendan (levosimendan) is for acute situations, although itself and length such as cardiac arrhythmia and increaseThe adverse events such as phase mortality are relevant. These tendencies have stoped it to be applied to chronic heart failure. Digoxin (Digoxin) is mouthTake cardiotonic, but its be limited to narrow therapeutic index, increase arrhythmogenic may and renal insufficiency in contraindication.

ADHF needs such therapy in heart failure urgently, its increase myocardial contractive power and do not have arrhythmogenic orMortality tendency, and can solve the huge unsatisfied needs of medical treatment of chronic heart failure.

Apelin is that previous orphan G-albumen-coupled receptor (GPCR), APJ are (also referred to as apelin acceptor, angiotensinsSample-1 acceptor, Angiotensin II sample-1 acceptor etc.) endogenic ligand. Apelin/APJ path is expressed in cardiovascular widelyIn system, and apelin has shown great useful Cardiovascular in preclinical models. The mankind's acute apelin usesCause periphery and crown vasodilation and increase cardiac output (Circulation.2010; 121:1818-1827). Therefore, APJThe exciting critical treatment target of suffering from patient in heart failure that just becoming. Think that the activation of apelin acceptor APJ increases cardiac muscle receiptsContracting power also provides Cardioprotective and without the unfavorable tendency of current therapy. But natural apelin shows extremely short half-life and workActing duration in body. Because quick serum is removed and through the proteolysis degraded of peptase effect, this is very short by halfThe phase of declining is the generally acknowledged main difficulty of sending this type of and treat endogenous peptide.

Current is to use heavy dose of interested treatment peptide to patient for overcoming a kind of method of this shortcoming, therebyEven if some treatment peptides are degraded, the still effective q.s for the treatment of. But the method is uncomfortable to patient. BecauseGreat majority treatments peptides can not be Orally administered, treatment peptide constantly infusion, by the frequent infusion of intravenous injection or by squareJust hypodermic injection approach is frequently used. Need to frequently use and also cause many potential peptide treatments to have unacceptable outstandingHigh treatment cost. The existence of the peptide of a large amount of degradeds also may produce undesirable side effect.

Use uncomfortable and expensive be why great majority have the treatment peptide of attractive biologically active characterCan not be developed to two reasons of drug candidates.

Therefore a kind of method of half-life that, extends peptide be slow down they degraded and still keep bioactive modeModified therapeutic peptide.

Therefore, qualification is simulated apelin function but is had the half-life of increase and show and naturally occurring apelin equivalenceOr more peptide and the polypeptide of high bioactivity are needed. In addition the conformational restriction that, qualification demonstrates increase (, is reached and ties upHold activity conformation state so that peptide and polypeptide can be interacted with its acceptor and/or other path target spot without extra foldOr reset ability) apelin analog peptide and polypeptide be needed. Other method comprises by peptide is conjugated to resistanceThe molecule that only they are removed by kidney reduces clearance rate.

Therefore, need to there is the treatment peptide of the modification of the half-life of increase, so that acting duration in longer body to be provided,Keep hypotoxicity simultaneously, retain the treatment advantage of the peptide of described modification.

Summary of the invention:

The present invention relates to by modifying interested treatment peptide or polypeptide is that APJ activator overcomes asking that in body, peptide is degradedTopic.

The object of this invention is to provide new peptide and polypeptide, its can be used as APJ activator and also have following be better than wildAt least one improvement of type apelin and other known apelin analog: the half-life of increase; To after using and/or after dissolvingThe larger immunity of degrading; And the conformational restriction increasing, it all shows the life identical or higher with wild type apelin simultaneouslyThing activity. Therefore, peptide of the present invention and polypeptide especially can be used for treatment or angiocardiopathy preventing, for example in heart failure the and heartIllness and the patient's condition of the illness that force failure is relevant and the patient's condition and the activation response to apj receptor activity.

In one embodiment, peptide of the present invention and polypeptide especially can be used for treatment or prevent with in heart failure relevantThe illness of illness or the patient's condition or the activation to apj receptor activity (or exciting) response. In another embodiment, peptide of the present inventionAnd polypeptide can be used for treating acute decompensation DHF (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillationMoving, Bu Lugedashi (Brugada) syndrome, Ventricular Tachycardia, atherosclerotic, hypertension, ISR, ischaemicProperty angiocardiopathy, cardiomyopathy, cardiac fibrosis, cardiac arrhythmia, water retention, diabetes (comprising GDM), obesityDisease, peripheral arterial disease, cerebrovas-cularaccident, temporary ischemic episode, traumatic brain injury, the ALS, (bag of burningDraw together sunburn) and pre-eclampsia.

The present invention relates to peptide and polypeptide, pharmaceutical composition and its production and use as described herein. Of the present inventionThe example of peptide and polypeptide comprises suc as formula the peptide described in any and polypeptide or its acid amides, ester, salt or its biology in I to IV and puting togetherThing and any peptide or the polypeptide specifically listed herein include but not limited to EXPERIMENTAL EXAMPLE.

Therefore the present invention provides peptide or the polypeptide of formula (I):

X1-R-P-R-X5-X6-X7-K-X9-P-X11-X12-X13

I

Wherein:

X1 is the N-end of this polypeptide, and does not have or be selected from Q, A and pE;

X5 is that L or X5 are selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form two sulphurKey;

X6 is S, s or a;

X7 is H, Aib or a; Or X7 is selected from C, c, hC and D-hC; The wherein side chain shape of the side chain of C, c, hC or D-hC and X12Become disulfide bond;

X9 is that G or X9 are selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form two sulphurKey;

Wherein in X5, X7 or X9 only one be selected from C, c, hC and D-hC;

X11 is D-Nle, Nle, M or f; And

X12 is selected from C, c, hC, D-hC; The wherein C of the side chain of C, c, hC or D-hC and X5, X7 or X9, c, hC or D-hCSide chain forms disulfide bond;

X13 is C-end, and does not have or be selected from (N-Me) F, F, f, a, y and Nal; Wherein:

Nle is L-nor-leucine;

D-hC is D-homocysteine

HC is L-homocysteine;

Nal is L-naphthylalanine;

Aib is 2-aminoisobutyric acid;

PE is L-Glutimic acid;

Or the acid amides of this polypeptide, ester or salt; Or the polypeptide of equal value with it substantially.

As further illustrated herein, with art-recognized 3 letters or 1 letter abbreviations represent to form peptide of the present invention andThe amino acid residue of polypeptide. Unless when above titled with " D ", otherwise amino acid is L-amino acid. When 1 letter abbreviations is capital letterWhen female, refer to L-amino acid. In the time that 1 letter abbreviations is lowercase, refer to D-amino acid.

Arbitrary listed amino acid residue above of formula I or its correlation described herein (for example, formula I to IV) can be to guardMode is substituted, prerequisite be peptide of the present invention or polypeptide still keep functional activity and structural property (for example, Increased Plasma Half-life, exempt fromBe subject to degraded, conformational restriction). Principle and the example of admissible conserved amino acid replacement will be further illustrated herein.

The present invention also provides bioconjugates or its polymer, and it comprises:

A. peptide or the polypeptide of any same form in formula I to IV,

B. the part of prolong half-life;

The part of wherein said peptide or polypeptide and described prolong half-life is or fusion covalently bound by connector optionally.

The part of prolong half-life of the present invention can merge, links, connects or be conjugated to peptide or polypeptide analog by covalency.The part of prolong half-life can be for example for example polyethylene glycol of polymer (PEG), cholesterol group, carbohydrate or oligomericSugar; Or with any natural or synthetic protein, polypeptide or the peptide of remedying (salvage) receptors bind. Preferably, extend and partly declineThe part of phase is optionally by covalently bound plasma proteins (albumin and the immune globulin to having long serum half-life of connectorIn vain). For example, the part of prolong half-life is IgG constant domain or its fragment (for example Fc district), human serum albumins (HSA) or whiteAlbumen-Binding peptide. Preferably, the part of the prolong half-life of bioconjugates is human serum albumins HuoFc district. Most preferablyGround, the part ShiFc district of the prolong half-life of bioconjugates.

The part of prolong half-life with the part that improves and/or do not disturb bioconjugates of the present invention for example thisThe mode of the biological function of bright peptide or polypeptide (formula I-IV) connects. In some embodiments, polypeptide of the present invention canOptionally merge the part to prolong half-life by connector. The part of prolong half-life can be that for example IgG of protein is constantTerritory or its fragment (for example Fc district), human serum albumins (HSA) or albumin-Binding peptide. Disclosed this proteinoid in literary compositionCan also form polymer.

In some embodiments, the part of prolong half-life (such as HSA, Fc etc.) is covalently bound or merge to formula I-IVIn the peptide of any same form or the N-end of polypeptide. In other embodiments, the part of prolong half-life (such as HSA, Fc etc.)Covalently bound or merge to the peptide of any same form or the C-end of polypeptide in formula I to IV of the present invention.

Polypeptide of the present invention or its bioconjugates, by activation apj receptor, can be used for treating the acute decompensation heartForce failure (ADHF), chronic heart failure, pulmonary hypertension, auricular fibrillation, Bu Lugeda Cotard, Ventricular Tachycardia,Atherosclerotic, hypertension, ISR, ischemic angiocardiopathy, cardiomyopathy, cardiac fibrosis, cardiac arrhythmia, waterRetention, diabetes (comprising gestational diabetes mellitus), obesity, peripheral arterial disease, cerebrovas-cularaccident, temporary ischemic episode, woundProperty brain damage, ALS, burn (comprising sunburn) and pre-eclampsia.

In a preferred embodiment, polypeptide of the present invention can be used for treating acute decompensation DHF(ADHF)。

In another embodiment, the present invention relates to for the individuality treatment in this treatment of needs, apj receptor be activatedThe illness of response or the method for disease, it comprises: give this individuality use effective dose according to the polypeptide of arbitrary formula in formula I to IV orIts acid amides, ester, salt or its bioconjugates, thus illness or the disease to apj receptor activation response of this individuality treated.

In yet another embodiment, the present invention relates to comprise according to the polypeptide of arbitrary formula in formula I to IV or its acid amides, ester,The pharmaceutical composition of salt or its bioconjugates and one or more pharmaceutically acceptable carrier.

In an embodiment again, the present invention relates to comprise according to the polypeptide of arbitrary formula in formula I to IV or its acid amides, ester,The combination product of the drug regimen of salt or its bioconjugates and one or more therapeutic activity agent.

In another embodiment, the present invention relates to activate the method for apj receptor in the individuality that has needs, it comprises:Give puting together according to the polypeptide of arbitrary formula in formula I to IV or its acid amides, ester, salt or its biology of this individuality administering therapeutic effective doseThing.

Detailed Description Of The Invention

For the object of explaining this description, except as otherwise noted, otherwise will be suitable for following definition, and in suitable feelingsUnder condition, the term using with odd number also can comprise plural number and vice versa.

As used herein, " illness or the disease of the regulation and control response to apj receptor ", " illness of the regulation and control response to APJ andThe patient's condition ", " illness and the patient's condition of the regulation and control response to apj receptor activity ", " activation (or exciting) response to apj receptor activityIllness " etc. term comprise acute decompensation DHF (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillationMoving, Bu Lugeda Cotard, Ventricular Tachycardia, atherosclerotic, hypertension, ISR, ischemic are cardiovascularDisease, cardiomyopathy, cardiac fibrosis, cardiac arrhythmia, water retention, diabetes (comprising gestational diabetes mellitus), obesity, peripheral arterialDisease, cerebrovas-cularaccident, temporary ischemic episode, traumatic brain injury, ALS, burn (comprising sunburn) and tendencyEclampsia.

As used herein, " activation of apj receptor activity " or " activation of apj receptor " refers to the increase of apj receptor activity.The activation of apj receptor activity, also referred to as " excitement " of apj receptor, for example, realizes by using peptide of the present invention and polypeptide.

As used herein, term " polypeptide " and " peptide " are used interchangeably, and refer to two or more ammonia that link togetherBase acid. Except the abbreviation of uncommon or alpha-non-natural amino acid listed in table 1 below, use 3 art-recognized lettersOr 1 letter abbreviations represent to form the amino acid residue of peptide of the present invention and polypeptide. Unless when above titled with " D ", otherwise ammoniaBase acid refers to L-amino acid. In the time that 1 letter abbreviations is capitalization, refer to D-amino acid. When 1 letter abbreviations is lowercaseTime, refer to L-amino acid. Represent peptide by group or character string or amino acid abbreviations. The left side that should point out peptide is N-end,And sequence is write from N-end to C-end.

Peptide of the present invention contain alpha-non-natural amino acid (, the non-existent compound of occurring in nature) and can alternatively use asOther amino acid analogue known in the art.

Some alpha-non-natural amino acid can be introduced by the technology of the following stated: Deiters etc., and JAmChemSoc125:11782-11783,2003; Wang and Schultz, Science301:964-967,2003; Wang etc., Science292:498-500,2001; Zhang etc., Science303:371-373,2004 or US patent No.7,083,970. In brief, thisSome in a little expression systems participate in directed mutagenesis, for example, send out thereby nonsense codon (amber type TAG) is introduced to code bookIn the ORFs of bright polypeptide. Then these expression vectors are introduced and can be utilized introduced nonsense codon specificityThe host of tRNA in, and load selected alpha-non-natural amino acid. To the object of other parts and conjugation of polypeptides of the present invention is hadThe special alpha-non-natural amino acid of benefit comprises those with acetylene and azido side chain.

Can be for example by adding chemical entities, such as carbohydrate group, bound phosphate groups, farnesyl-, different farnesyl-,Fatty acid group, put together with connector, functionalized or other modify etc. modify one or more in peptide of the present invention natural orAlpha-non-natural amino acid. These modifications can realize with locus specificity or non-locus specificity mode. In a preferred embodimentIn, the modification of peptide produces more stable peptide (for example, showing the peptide of longer Half-life in vivo). These modifications can comprise introducing volumeOuter D-amino acid etc. These modify the required biologically active that all can not disturb in fact peptide, and it is desired to give this peptideCharacter, for example, the biologically active of enhancing.

With respect to wild-type protein, described modification has strengthened the biological property of albumen of the present invention, and in some casesAs the tie point of for example mark and the agent of albumen Increased Plasma Half-life, and for these variants being linked to the table of solid carrierFace.

In certain embodiments, for for example prolong half-life or improve the biological property of these polypeptide and/or peptideObject, use these to modify (such as, site-specific sex modification) and connect conjugate (for example, PEG group) to of the present inventionOn polypeptide and/or peptide. To further set forth these technology herein.

In other embodiments, these modify (for example, site-specific sex modification) for connecting prolongation polypeptide of the present inventionOther polymer and little molecule and the recombinant protein sequence of half-life. This type of embodiment comprises connection aliphatic acid or spyOpposite sex albumin binding compounds is to polypeptide and/or peptide. In other embodiments, these are modified at specific amino acids typeOn carry out and can be connected in one or more site on polypeptide.

In other embodiments, these modify (for example, site-specific sex modification) as producing wild type and/or variantThe connection means of polymer (for example, dimer (homodimer or heterodimer) or tripolymer or the tetramer). These are manyGlycoprotein polyprotein precursor molecule can have extraly through amino terminal or carboxyl terminal and is connected or fused to other albumen (for example Fc, human bloodPure albumen (HSA) etc.) group such as PEG, sugar and/or PEG-cholesterol conjugate etc.

In other embodiments, these site-specific sex modifications are for the preparation of albumen, polypeptide and/or peptide, wherein siteSpecificity is mixed amino acid (p-acetyl group-Phe, p-that pyrrolysine or pyrrolysine analog or non-natural existAzido-Phe) position allow controlled directional and connect these albumen, polypeptide and/or peptide to the surface of solid carrier, orAllow to connect the group such as PEG, sugar and/or PEG-cholesterol conjugate.

In other embodiments, these site-specific sex modifications for locus specificity crosslinking protein, polypeptide and/orPeptide, forms oligomeric thing thus, includes but not limited to heterodimer and heterotrimer. In other embodiments, thisA little site-specific sex modifications, for locus specificity crosslinking protein, polypeptide and/or peptide, form protein-protein conjugate, egg thusIn vain-polypeptide conjugate, protein-peptide conjugate, polypeptide-polypeptide conjugate, polypeptide-peptide conjugate or peptide-peptide conjugate. At otherIn embodiment, the specific modification in site can comprise that branch point is to allow more than one types of molecules to be connected in albumen, polypeptide or peptideSingle site.

In other embodiments, modification listed herein can realize in non-locus specificity mode, and producesProtein-protein conjugate of the present invention, albumen-polypeptide conjugate, protein-peptide conjugate, polypeptide-polypeptide conjugate, polypeptide-peptideConjugate or peptide-peptide conjugate.

In some embodiments, the invention provides comprise at least one be bonded to antibody (for example specific binding as thisThe antibody of literary composition disclosed peptide or polypeptide) formula I to IV in the peptide of arbitrary formula or the compound of polypeptide.

It will be understood by a person skilled in the art that, can in the sequence of arbitrary polypeptide described herein, carry out different 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors(for example, conserved amino acid replaces), and must not reduce its activity. As used herein, " be typically used as the amino of its substituentAcid " comprise and guard replacement (, being there is the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of comparable chemical characteristic). For the conservative object replacing, the non-utmost pointProperty (hydrophobicity) amino acid comprises alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, look ammoniaAcid and methionine. Polarity (hydrophily) neutral amino acid comprises serine, threonine, cysteine, tyrosine, asparagineAnd glutamine. Positively charged (alkalescence) amino acid comprises arginine, lysine and histidine. Electronegative (acidity) amino acidComprise aspartic acid and glutamic acid. The example of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor comprises with its corresponding L-amino acid of D-49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, with high by halfCystine or other have containing the alpha-non-natural amino acid of thiol side chain and replace cysteine, with high-lysine, DAB, twoAlanine, ornithine or other have containing the alpha-non-natural amino acid of amino side chain and replace lysine, or replace with norvalineAlanine etc.

Term used herein " amino acid " refer to naturally occurring amino acid, alpha-non-natural amino acid, amino acid analogue andThe amino acid analog thing working to be similar to naturally occurring amino acid whose mode, if its structure allows D and L alloisomerismBody, all with its D and L stereoisomer. Herein by its title, their known 3 letter characters or pass through IUPAC-IUB1 letter character that BiochemicalNomenclatureCommission (biochemical nomenclature commission) recommends representsAmino acid.

Term " naturally occurring " refers at occurring in nature and finds and without the material of artificial treatment. Similarly, this paper instituteRefer to not at occurring in nature and find or through mankind's structural modification or synthetic thing with " non-natural exists ", " non-natural " etc.Matter. When with amino acid coupling, term " naturally occurring " relates to 20 kinds of conventional amino acid (, alanine (A or Ala), half GuangPropylhomoserin (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly),Histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M orMet), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (SOr Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp) and tyrosine (Y or Tyr)).

Term used herein " alpha-non-natural amino acid (non-naturalaminoacid) " and " alpha-non-natural amino acid(unnaturalaminoacid) " be used interchangeably, expression cannot be used from the not modified of arbitrary organism or through repairingThe amino acid structure producing in biosynthesis mode in gene (no matter identical or different) organism in office of decorations. These termsRefer to non-existent amino acid residue in natural existence (wild type) apelin protein sequence or sequence of the present invention. These compriseBut be not limited to not to be the modified amino acid of one of 20 kinds of naturally occurring amino acid and/or amino acid analogue, seleno half(Pcl is for example described in the open WO2010/ of PCT patent for cystine, pyrrolysine (Pyl) or pyrroline-carboxy-lysine48582). This type of alpha-non-natural amino acid residue can be by replacing naturally occurring amino acid and/or passing through alpha-non-natural amino acidInsert in natural existence (wild type) Apelin protein sequence or sequence of the present invention and introduce. Also alpha-non-natural amino acid can be mixed residualBase, to give apelin molecule desired function, for example, connects functional moiety's (for example, PEG) ability. When with amino acid couplingTime, symbol " U " means " alpha-non-natural amino acid " used herein.

In addition, should be understood that these " alpha-non-natural amino acids " need through the tRNA of modification and synthetic through the tRNA modifyingEnzyme (RS) is incorporated in albumen. The orthogonal tRNA/RS of these " selections " is to passing through as the system of selection of people's exploitations such as SchultzOr produce by random or target sudden change. For example, pyrroline-carboxy-lysine is " natural amino acid ", because it is to pass throughBe converted into the gene host cell from an organism, produce in biosynthesis mode, and because it is by using skySo tRNA and tRNA synthase gene and be incorporated in albumen, and p-aminophenyl alanine (referring to, Generationofabacteriumwitha21aminoacidgeneticcode,MehlRA,AndersonJC,SantoroSW,WangL,MartinAB,KingDS,HornDM,SchultzPG.JAmChemSoc.2003Jan29；125(4):Be 935-9) " alpha-non-natural amino acid ", although because produce in biosynthesis mode, it is the orthogonal tRNA/ by " selection "TRNA synzyme is to being incorporated in albumen.

Modified coded amino acid includes but not limited to hydroxy-proline, gamma carboxyglutamate, O-phosphoric acid silk ammoniaAcid, azetidine formic acid, AAA, 3-aminoadipic acid, Beta-alanine, alanine, 2-amino-butyric acid, 4-ammoniaBase butyric acid, 6-aminocaprolc acid, 2-aminoheptylic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-diaminopimelic acid, the tert-butyl group are sweetPropylhomoserin, 2,4-diaminourea isobutyric acid, desmosine, 2,2 '-diaminopimelic acid, 2,3-diaminopropionic acid, Ethylglycocoll, N-Methylglycine, N-ethyl asparagine, high proline, oxylysine, not-oxylysine, 3-hydroxy-proline, 4-hydroxylBase proline, isodensmosine, not-isoleucine, N-methylalanine, sarcosine, N-methyl isoleucine, N-methylAmyl group glycine, N-methylvaline, naphthylalanine, norvaline, nor-leucine, ornithine, amyl group glycine, pipecolinic acidAnd Thioproline. Term " amino acid " is also included as the metabolin in some organism, but without for mixing albumenThe naturally occurring amino acid of genetic code coding. These amino acid include but not limited to ornithine, D-Orn and D-essence ammoniaAcid.

Term used herein " amino acid analogue " refers to have the basic chemistry knot identical with naturally occurring amino acidThe compound of structure (for example, being bonded to the α-carbon of hydrogen, carboxyl, amino and R group). Amino acid analogue comprises by reversibly or notReversibly chemical sealing or its C-terminal carboxyl group, its N-terminal amino group and/or its side chain functional group are natural through chemical modificationAnd alpha-non-natural amino acid. These analogs include but not limited to methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-half GuangPropylhomoserin, S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteine sulfone, aspartic acid-(Beta-methyl ester), N-ethylGlycine, alanine formamide, homoserine, nor-leucine and methionine methyl sulfonium.

Table 1: alpha-non-natural amino acid as described in the present invention:

Nal refers to 1-naphthylalanine and 2-naphthylalanine, preferably 2-naphthylalanine.

As used herein, term " acid amides " refers to amide derivatives (for example ,-C (O) NH of C-terminal carboxylic acid group₂、-C(O)NH-C_1-6Alkyl ,-C (O) NH-C_1-2Alkyl phenyl ,-C (O) NH-NHBn ,-C (O)-4 Phenoxypiperidines or-C (O) N (C_1-6Alkyl)₂)。

Term " acid amides " also refers to derivative (for example ,-NHC (O) C of N-terminal amino group_1-16Alkyl ,-NHC (O) (CH₂)_nPh(n is 1 to 6 integer) ,-NHC (O) (CH₂)₂CO₂H、4-Cl-Ph-(CH₂)₃C(O)NH-、C₁₁H₂₃C(O)NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-C(O)-NH-、C₁₃H₂₇C(O)NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-C(O)-NH-；

C₁₅H₂₇C(O)NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-C(O)NH-、Ph-CH₂CH₂NHC (O)-NH-or CH₃(OCH₂CH₂)_mC (O) NH-(m is 1 to 12 integer).

As used herein, term " ester " refers to the ester derivant (for example-COOR) of C-terminal carboxylic acid group, wherein this esterR refers to C_1-6Alkyl, such as methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl etc.; C_3-8Cycloalkyl, such as cyclopenta, cyclohexylDeng; C_6-10Aryl, such as phenyl, Alpha-Naphthyl etc.; C_6-10Aryl-C_1-6Alkyl, for example phenyl-C_1-2Alkyl, such as benzyl, benzene secondBase, benzhydryl etc.; And Alpha-Naphthyl-C_1-2Alkyl, such as Alpha-Naphthyl methyl etc. That also can mention is valeryl oxygen Ji JiaBase esters etc., it is typically used as Orally administered ester. When polypeptide of the present invention has extra carboxyl in the position except C-endOr when carboxylate group, these groups are amidated or the those polypeptides of esterification also falls in the scope of polypeptide of the present invention. At thisPlant under situation, these esters for example belong to the ester of identical type with C-terminal ester referred to above.

Term alkyl refer to comprise 1 to 20 carbon atom complete saturated branch or unbranched (or straight chain or lineProperty) hydrocarbyl group. Preferably, alkyl comprises 1 to 7 carbon atom, and more preferably 1 to 4 carbon atom.

Term aryl refers to aromatic hydrocarbon group monocycle or two rings at loop section with 6-10 carbon atom. ArylRepresentative example is phenyl or naphthyl.

Term heteroaryl comprises monocycle or bicyclic heteroaryl, its contain 5-10 be selected from carbon atom and 1 to 5 heteroatomicRing members, and each hetero atom is independently selected from O, N or S, and wherein S and N can be oxidizing to the different states of oxidation. For twoRing heteroaryl system, this system is completely fragrant (all rings are all fragrant).

Term cycloalkyl refers to the saturated or unsaturated but non-of 3-12 carbon atom, preferably 3-8 or 3-7 carbon atomThe monocyclic, bicyclic or tricyclic hydrocarbyl group of fragrance. For two rings and tricyclic naphthenes based system, all rings are all non-aromatic.

Term heterocyclic radical refers to saturated or unsaturated non-aromatic (part is undersaturated) ring, its be 4-, 5-, 6-or7-unit monocycle, and contain at least one and be selected from the hetero atom of O, S and N, wherein N and S can also optionally be oxidizing to different oxygenChange state. In one embodiment, heterocyclic radical group represents to contain 5-7 annular atoms and optionally contains other and is selected from O, SOr the heteroatomic saturated monocycle of N.

Term " APJ " (also referred to as " apelin acceptor ", " angiotensins sample-1 acceptor ", " Angiotensin II-sample 1 is subject toBody " etc.) represent that the Gi with 380 residues, 7 membrane spaning domains of the assignment of genes gene mapping on Human chromosome 11 long-armed is evenConnection acceptor (NCBI reference sequences number: NP_005152.1, and by NCBI reference sequences number: NM_005161 coding). APJ is 1993Year use degeneracy oligonucleotide primer from genome human DNA clone first obtain (O'Dowd etc., Gene, 136:355-60,1993) and with 1 type angiotensin-ii receptor there is remarkable homology. Although there is this homology, but, angiotensinsII is not in conjunction with APJ. Although many year as orphan, isolated endogenic ligand called after apelin (Tatemoto etc.,BiochemBiophysResCommun251,471-6(1998))。

Term " apelin ", represent have 77 residues front albumen (NCBI reference sequences number: NP_0059109.3, andBy NCBI reference sequences number: NM_017413.3 coding), it is processed into the biologically active form of apelin peptide, such asApelin-36, apelin-17, apelin-16, apelin-13, apelin-12. Total length mature peptide is also referred to as " apelin-36 ", comprise 36 amino acid, but the pyroglutamic acid form of 13 bodies (apelin-13) that the most efficient hypotype is apelin, its quiltBe called " Pyr-1-apelin-13 or Pyr¹-apelin-13 ". Different apelin forms are for example described in United States Patent (USP) 6,492,In 324B1.

Term " conjugate " and " bioconjugates " are used interchangeably, and mean many by any same form in formula I to IVOptional by the entity of the covalently bound formation of connector between peptide and the part of prolong half-life. Term " conjugate " or " biologyConjugate " be also intended to comprise between APJ agonist polypeptide or the polypeptide of formula I, II, III or IV and the part of prolong half-life and meltClose the entity of formation.

The part of term prolong half-life can by covalently bound/link or merge to peptide or polypeptide analog. Extend halfThe part of phase of declining may be for example for example polyethylene glycol of polymer (PEG), cholesterol group, carbohydrate or compound sugar; WithRemedy any natural or synthetic protein, polypeptide or the peptide of (salvage) receptors bind. Preferably, the portion of prolong half-lifeDivide optional by the covalently bound plasma proteins (albumin and immunoglobulin (Ig)) to thering is long serum half-life of connector. ExampleAs, the part of prolong half-life is IgG constant domain or its fragment (for example Fc district), human serum albumins (HSA) or albumin-knotClose polypeptide. Most preferably, the part ShiFc district of the prolong half-life of bioconjugates.

Term " increase half-life " or " increase serum half-life " or " prolong half-life " mean with respect to its unmodifiedForm (or naked form of peptide), the positivity of the circulating half-life of the biologically active molecules (for example apelin13) of modification changes.By taking blood sample and measure the dense of molecule described in each sample in the different time points of using after biologically active moleculesDegree is measured serum half-life. The serum-concentration of measuring in time changes the molecule (molecule of for example puting together) that allows to calculate modificationSerum half-life. By by modify the serum half-life of molecule (molecule of for example puting together) and the molecule of unmodified (for exampleApelin13) compare, can measure the relative increase of serum half-life or t1/2. Described increase is desirably at least aboutTwice, but less increase may be useful.

Polypeptide of the present invention:

This paper describes each embodiment of the present invention. It should be understood that the feature described in each embodiment canWith other Feature Combination indicating, thereby provide other embodiment.

In embodiment 1, therefore the present invention provides peptide or the polypeptide of formula (I):

X1-R-P-R-X5-X6-X7-K-X9-P-X11-X12-X13

I

Wherein:

X1 is the N-end of this polypeptide, and does not have or be selected from A, Q and pE;

X6 is S, s or a;

Wherein in X5, X7 or X9 only one be selected from C, c, hC and D-hC;

X11 is D-Nle, Nle, M or f; And

X12 is selected from C, c, hC, D-hC; The wherein C of one of the side chain of C, c, hC or D-hC and X5, X7 or X9, c, hC or D-The side chain of hC forms disulfide bond;

Nle is L-nor-leucine;

D-hC is D-homocysteine

HC is L-homocysteine;

Nal is L-naphthylalanine;

Aib is 2-aminoisobutyric acid;

PE is L-Glutimic acid;

In embodiment 2, therefore the present invention provides peptide or the polypeptide of formula (II):

X5 is selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form disulfide bond;

X6 is S, s or a;

X7 is H, Aib or a;

X11 is D-Nle, Nle, M or f;

X12 is selected from C, c, hC, D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X5 form disulfide bond;

X13 is C-end, and does not have or be selected from (N-Me) F, F, f, a, y and Nal;

In embodiment 2A, the invention provides peptide or the polypeptide of formula II, wherein:

X1 is the N-end of this polypeptide, and does not exist or pE;

X6 is S, s or a;

X7 is H, Aib or a;

X11 is D-Nle, Nle or f;

In embodiment 3, the present invention relates to peptide or the polypeptide of formula III:

X6 is S, s or a;

X7 is H, Aib or a;

X9 is selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form disulfide bond;

X11 is D-Nle, Nle, M or f; And

X12 is selected from C, c, hC, D-hC; Wherein the side chain of the side chain of C, c, hC or D-hc and the C of X9, c, hC or D-hC formsDisulfide bond;

In embodiment 3A, the invention provides peptide or the polypeptide of formula III, wherein:

X1 is the N-end of this polypeptide, and does not exist or pE;

X6 is S, s or a;

X7 is H, Aib or a;

X11 is D-Nle, Nle or f; And

In embodiment 4, the invention provides peptide or the polypeptide of formula IV:

X6 is S, s or a;

X7 is selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form disulfide bond;

X11 is D-Nle, Nle, M or f; And

X12 is selected from C, c, hC, D-hC; Wherein the side chain of the side chain of C, c, hC or D-hc and the C of X7, c, hC or D-hC formsDisulfide bond;

In embodiment 4A, the present invention relates to peptide or the polypeptide of formula IV, wherein:

X1 is the N-end of this polypeptide, and does not exist or pE;

X6 is S, s or a;

X11 is D-Nle, Nle or f; And

In embodiment 5, the present invention relates to according to the polypeptide described in any one in embodiment 1,2,3 and 4, whereinX1 is pE; Or the acid amides of this polypeptide, ester or salt.

In embodiment 5A, the present invention relates to according to the polypeptide described in any one in embodiment 1,2,3 and 4, itsMiddle X1 is A or Q; Or the acid amides of this polypeptide, ester or salt. This embodiment one special aspect, A and Q chemistry are connected to prolongationThe part of half-life.

In embodiment 6, the present invention relates to according to the polypeptide described in any one in embodiment 1 to 4, wherein X1 is notExist; Or the acid amides of this polypeptide, ester or salt.

In embodiment 7, the present invention relates to according to the polypeptide described in any one in embodiment 1 to 4 and 6, whereinN-end is acid amides; Or the salt of this polypeptide.

In embodiment 8, the present invention relates to according to the polypeptide described in embodiment 7, wherein N-end Shi Shi – NHRAcid amides, and R is acetyl group, benzoyl, phenacyl, succinyl group, caprylyl, 4-phenyl bytyry, 4-Cl-Ph-(CH₂)₃C (O)-or Ph-CH₂CH₂NHC (O)-; Or the salt of this polypeptide.

In embodiment (8A), the present invention relates in formula I to IV any one or above-described any other class and(according in embodiment 1 to 8 described in any one) peptide and polypeptide in subclass described in any one, wherein N-end isAcid amides and the R1 of Shi – NHR1 are CH₃C(O)-、CH₃-(O-CH₂CH₂)_m-C (O)-, palmityl (O2Oc)_p, myristoyl(O2Oc)_p, lauroyl (O2Oc)_pOr Ph-CH₂CH₂NHC (O)-, benzoyl, phenacyl, succinyl group, caprylyl, 4-Phenyl bytyry, 4-Cl-Ph-(CH2)₃C (O)-or Ph-CH₂CH₂NHC (O)-; And wherein

P is integer 1 to 4;

M is integer 1 to 12;

Lauroyl (O2Oc)_pC₁₁H₂₃C(O)[NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-C(O)]_p-；

Myristoyl (O2Oc)_pC₁₃H₂₇C(O)[NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-C(O)]_p-；

Palmityl (O2Oc)_pC₁₅H₃₁C(O)[NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-C(O)]_p-, or the salt of this peptide. N-The example of terminal amide has been described in the US provisional application No.61/591 submitting on January 27th, 2012,557 (attorney docketPAT054961-US-PSP), in, be introduced into as a reference.

In embodiment 9, the present invention relates to according to the polypeptide described in any one in embodiment 1 to 8A, wherein X13F or f; Or the acid amides of this polypeptide, ester or salt.

In embodiment 10, the present invention relates to according to the polypeptide described in any one in embodiment 1 to 8A, whereinX13 does not exist; Or the acid amides of this polypeptide, ester or salt.

In embodiment 11, the present invention relates to according to the polypeptide described in any one in embodiment 1 to 10, wherein C-End is acid amides; Or the salt of this polypeptide.

In embodiment 12, the present invention relates to according to the polypeptide described in embodiment 11, wherein C-end Shi Shi – C(O) acid amides and the R2 of-R2 are-NH₂,-NH-Me ,-NH-NHBn or-NH-(CH₂)₂-Ph; Or the salt of this polypeptide.

In embodiment 13, the present invention relates to according to the polypeptide described in any one in embodiment 1-12, wherein X6S; Or the acid amides of this polypeptide, ester or salt.

In embodiment 14, the present invention relates to according to the polypeptide described in any one in embodiment 1-13, wherein X7H; Or the acid amides of this polypeptide, ester or salt.

In embodiment 15, the present invention relates to according to the polypeptide described in any one in embodiment 1 to 14, whereinX11 is Nle or D-Nle; Or the acid amides of this polypeptide, ester or salt.

In embodiment 15A, the present invention relates in embodiment 1 to 17 polypeptide described in any one, Qi ZhongyouThe C-end of X11-X12-X13 part composition be selected from Nle-C*-F, Nle-(D-hC*)-F, Nle-(hC) * F, Nle-c*-F,Nle-C*-f, Nle-C*-f, Nle-C* phenyl ethylamine and (D-Nle) C*-y, or the salt of this polypeptide.

In an embodiment 15B, the present invention relates in embodiment 1 to 15 peptide or the polypeptide of any one, whereinIn amino acid X1, X5, X6, X7, X9 and X11 to X13 at least two with Pyr-1-apelin-13 in the corresponding amino acid that existsDifferent. In another embodiment, the present invention relates in embodiment 1 to 15 peptide or the polypeptide of any one, wherein amino acidX1, X5, X6, X7, X9 are different from the corresponding amino acid existing in Pyr-1-apelin-13 with in X11 to X13 at least three.In another embodiment, the present invention relates in embodiment 1 to 15 peptide or the polypeptide of any one, wherein amino acid X1, X5,X6, X7, X9 are different from the corresponding amino acid existing in Pyr-1-apelin-13 with in X11 to X13 at least four.

In another embodiment, X1, X5, X6, X7, X9 and X11 to X13 amino acid are by example part belowX1, X5, X6, X7, X9 and X11 to X13 amino acid defined those.

In another embodiment, individual polypeptide of the present invention is those or its pharmacy of listing in embodiment part belowUpper acceptable salt.

Except as otherwise noted, otherwise term " polypeptide of the present invention " refers to formula (I) and minor (formula I, II, III or IV) thereofPolypeptide; Or its acid amides, ester or salt.

Except as otherwise noted, otherwise term " polypeptide of the present invention ", " peptide of the present invention ", " apelin peptide agonists " etc. bePeptide and the polypeptide of finger formula I and minor thereof (formula I, II, III or IV); Or its acid amides, ester or salt. Peptide of the present invention and polypeptide showGo out with known apelin peptide as herein described and polypeptide and (include but not limited to wild type apelin, apelin-13 and pyr-1-Apelin-13) of equal value or improved activity and/or plasma stability substantially.

Peptide of the present invention and polypeptide also comprise with according to the peptide of arbitrary formula in formula I, I, II, III or IV and polypeptide or its acylAmine, ester or salt and with any peptide that herein (including but not limited to EXPERIMENTAL EXAMPLE) specifically lists or polypeptide have at least aboutThe peptide of 95% homogeneity and polypeptide.

As used herein, phrase " homologous amino acid sequence " or its version refer to so that few prescribed percentage at ammoniaThe sequence that homology on base sour water is flat is feature, and can exchange and use with " sequence homogeneity ". Homologous amino acid sequence comprisesThose contain conserved amino acid and replace, and polypeptide has identical combination and/or active amino acid sequence. In some embodimentsIn, if amino acid sequence and comparative sequence have at least 60% or higher, high to 99% homogeneity, it is homology.In some embodiments, if amino acid sequence and comparative sequence share one or more, 60 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors of as many as, interpolationOr disappearance, it is homology. In some embodiments, homologous amino acid sequence has and is no more than 5 or be no more than 3 guarantorsKeep 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

Homology also can be in polypeptide level. Peptide of the present invention or polypeptide or its part and different aminoacids sequence sameProperty degree or percentage calculate as follows: by the number of exact matching in two sequence alignments divided by " sequence of the present invention " orThe shortest person's length in " exogenous array ". Results expression is homogeneity percentage.

Comprise with amino acid sequence described in specific embodiment and there is about 80-99.9%, preferred 90-99.9% homogeneityAmino acid sequence, and the polypeptide with the plasma stability that is better than apelin-13 or pyr-1-apelin-13 to fall into the present invention manyIn the scope of peptide. In one embodiment, plasma stability improves at least 2 times. In one embodiment, polypeptide tool of the present inventionThere is the plasma stability of at least 30 minutes. In another embodiment, polypeptide of the present invention has at least 60 minutes or at least 80Minute, preferably at least 100 minutes and the more preferably plasma stability of at least 150 minutes.

Term " substantially of equal value " means that the character such as acceptor-combination is active, signal transduction is active are of equal value. Therefore,Can allow even to there are differences such as the grade of the intensity of receptor-binding activity and the molecular weight of polypeptide.

Polypeptide or its are by replacing, lack, add or inserting that one or more is amino acid whose substantially of equal value as described hereinThing can be described as the basic equivalent of the polypeptide that contains amino acid sequence of above meaning. Polypeptide or 1 are to 5 as described hereinIndividual, preferably 1 to 3 and more preferably 1 or 2 amino acid can by its basic equivalent natural or that alpha-non-natural amino acid replacesBe called the basic equivalent of the polypeptide that contains amino acid sequence of above meaning. Other modifications and change can comprise uses D-amino acidSubstitute L-amino acid, or other variations include but not limited to phosphorylation, carboxylation, alkylation etc., as long as maintain formula I, IA, II, IIIOr the apelin agonist activity of the peptide of IV or polypeptide, and plasma stability is improved and is better than the burnt paddy ammonia of apelin-13Acidifying form.

In embodiment 17, the invention still further relates to bioconjugates or its polymer, it comprises:

A. according to the peptide of any one formula I, II, III or IV in foregoing embodiments or polypeptide, its acid amides, salt or ester;

B. the part of prolong half-life;

In embodiment 17A, the part of prolong half-life is optionally covalently bound by connector part or merge to formulaThe N-end of the peptide of I, II, III or IV.

In embodiment 17B, the part of prolong half-life is optionally covalently bound by connector part or merge to formulaThe C-end of the peptide of I, II, III or IV.

In embodiment 17C, the part of prolong half-life is covalently bound or merge to the peptide of formula I, II, III or IVSide chain, the part of for example prolong half-life is optionally connected to K, Orn, Dab, Dap, hK or 4-amino-lsn by connector partThe amino group of side chain. Preferably, the part of prolong half-life optionally by connector part be connected to formula I, II, III orThe N-end of the peptide of IV.

In embodiment 18, the present invention relates to according to the bioconjugates described in embodiment 17 or its polymer, itsThe part of middle prolong half-life is IgG constant domain or its fragment or human serum albumins.

In embodiment 19, the present invention relates to according to the bioconjugates of embodiment 17 or 18, wherein extend and partly declineThe part of phase is the Fc fragment with the FcLALA modification of LALA sudden change (L234A, L235A).

In embodiment 20, the present invention relates to according to the bioconjugates of embodiment 19, wherein prolong half-lifePart is Fc domain, and its polypeptide by connector and formula I, II, III or IV merges, and under wherein connector hasFormula :-[GGGGS] n-, n is 2 or 3, and the polypeptide of formula I, II, III or IV contains naturally occurring amino acid.

In embodiment 21, the present invention relates to according to the bioconjugates of embodiment 20, wherein polypeptide be selected fromUnder the polypeptide of formula I: QRPRC*SHKGPMC*F, QRPRLSHKC*PMC*F and QRPRLSC*KGPMC*F, wherein use " * " markTwo amino acid represent to form by their side chain the amino acid of disulfide bond or amido link.

In embodiment 22, the present invention relates to according to the bioconjugates described in embodiment 17 or 18 or its polyBody, wherein the part of prolong half-life is human serum albumins.

In embodiment 23, the present invention relates to according to the bioconjugates of embodiment 22, wherein human serum albuminsBe connected to the N-end of the polypeptide of any one in formula I to IV by the connector chemistry of following formula:

Wherein x is 1-20, and R is alkylidene, cycloalkyl, aryl or heteroaryl linear or side chain or its combination, and R ' isAlkylidene, aryl or cycloalkyl or its combination linear or side chain.

In embodiment 24, the present invention relates to according to the bioconjugates of embodiment 17 or 18, wherein human seralbuminAlbumen is connected to the C-end of the polypeptide of any one in formula I to IV by the connector chemistry of following formula:

The part of prolong half-life

The part of prolong half-life of the present invention can link, connect, put together or merge to peptide or polypeptide analog by covalency.The part of prolong half-life may be for example for example polyethylene glycol of polymer (PEG), cholesterol group, carbohydrate or oligomericSugar; With any natural or synthetic protein, polypeptide or the peptide of remedying (salvage) receptors bind. Preferably, prolong half-lifePart optionally by the covalently bound plasma proteins (albumin and immunoglobulin (Ig)) to there being long serum half-life of connector.For example, the part of prolong half-life be IgG constant domain or its fragment (for example Fc district), human serum albumins (HSA) or albumin-Binding peptide. Preferably, the part of the prolong half-life of bioconjugates is human serum albumins HuoFc district.

The part of prolong half-life comprises albumin, and the protein that it refers to maximum in blood plasma has for monomer whose shapeThe molecular weight of about 65 to 67 kilodaltons of formula, this depends on source of species. Term " albumin " can with " seralbumin "Exchange and use, do not mean that and limit the albuminous source that forms conjugate with the peptide of modification of the present invention. Therefore, in literary composition, useTerm " albumin " can refer to from the albumin of for example blood of natural source or serum solution purifying, or it can relate to chemical synthesisOr restructuring produce albumin. The peptide of modification of the present invention or polypeptide are preferably optionally fastened and are connected to albuminous table by connectorThe free sulfhydryl groups of cysteine-34 on face.

The part of prolong half-life comprises " natural Fc ", and it refers to and comprises by digesting complete antibody produces or other sideMolecule or the sequence of the non--sequence of antigen-binding fragment (monomer or polymer form) that formula produces, and may be containing hingedSequence. Preferably people source, the original immunoglobulin (Ig) source of natural Fc, and can be any immunoglobulin (Ig), although IgG1With IgG2 be preferred. Natural Fc molecule is by being connected to dimer by covalency (being disulfide bond) and non-covalent connectionOr the monomer polypeptide of polymer form forms. The number of the intermolecular disulfide bond between the monomer subunit of natural Fc molecule be 1 to4, this depends on classification (for example IgG, IgA and IgE) or subclass (for example IgG1, IgG2, IgG3, IgA1 and IgGA2). NaturalAn example of Fc be the disulfide bonding that produced by the papain digestion of IgG dimer (referring to people such as Ellison,1982, NucleicAcidsRes.10:4071-9). The term " natural Fc " that uses in literary composition summarize refer to monomer, dimer andPolymer form.

The part of prolong half-life comprises " Fc variant ", its refer to modified by natural Fc but still comprise and remedy acceptorMolecule or the sequence of FcRn (neonatal Fc receptor) binding site. International publication number .WO97/34631 and WO96/32478 retouchStated exemplary Fc variant and with the interaction of remedying acceptor, be hereby incorporated by. Therefore, term " Fc variant " canCan comprise from the humanized molecule of the natural Fc of non-human or sequence. In addition, natural Fc comprises the region that can be removed, because ofFor they provide the unwanted architectural feature of bioconjugates of the present invention or biologically active. Therefore, term " Fc variant " comprisesSuch molecule or sequence, they lack one or more natural Fc sites or residue or one or more Fc sites or residual whereinBase is modified, and described site or residue affect or relate to (1) disulfide bond and form, the incompatibility of the host cell of (2) and selection,(3) once the heterogeneity of expressing N-end in the host cell of selecting, (4) glycosylation, (5) interact with complement, (6)With Fc receptors bind not with remedy receptors bind, or (7) ADCC (ADCC). Fc variant enters hereinafterOne step is described in detail.

The part of prolong half-life relates to " the Fc domain " that comprise natural Fc defined above and Fc variant and sequence.As Fc variant and natural Fc molecule, term " Fc domain " comprise the monomer that produced by digestion complete antibody or alternate manner orThe molecule of polymer form. In some embodiments of the present invention, Fc domain can pass through for example Fc domain and peptide orderCovalent bond between row is conjugated to the polypeptide of any same form in formula I ' or formula I-IV. This type of Fc albumen can pass through Fc domainConnect and form polymer, and the polymer of these Fc albumen and they is one aspect of the present invention.

The part of prolong half-life comprises " the Fc fragment of modifying ", and it means the Fc sheet of the antibody of the sequence that comprises modificationSection. Fc fragment is the antibody moiety that comprises CH2, CH3 and part hinge area. The Fc fragment of modifying may available from IgGl for example,IgG2, IgG3 or IgG4. FcLALA is the Fc fragment with the modification of LALA sudden change (L234A, L235A), its effect to reducePower triggers ADCC, and the combination of weak ground and activation people complement. The people 2007Nature449:101-104 such as Hessell. To Fc sheetThe other modification of section is described in for example U.S. patent No.7, in 217,798.

The term " polymer " of the molecule that is applied to Fc domain or comprise Fc domain refers to have two of covalency connectionThe molecule of individual or multiple polypeptide chains. For example IgG molecule typically forms dimer, and therefore comprises the IgG molecule of dimerizationBioconjugates is by the polypeptide chain merging to two formula I, IA, II, III or IV.

Connector

Any connector group is optional. In the time existing, its chemical constitution is not critical because its mainly asSpacerarm.

Connector is the chemical part that contains two reactive group/functional groups, one of them reactive group/functional groupCan react with polypeptide, another can with the partial reaction of prolong half-life. Two reactive groups of connector are by connectingConnect group and connect, the structure of linking group is not critical, as long as it does not disturb the portion of connector and peptide and prolong half-lifeThe coupling dividing.

Connector may be by the Amino acid profile linking together by peptide bond. In some embodiments of the present invention,Connector is by 1 to 20 Amino acid profile connecting by peptide bond, and wherein amino acid is selected from 20 kinds of naturally occurring amino acid. ?In each embodiment, described 1 to 20 amino acid be selected from amino acid glycine, serine, alanine, proline, asparagine,Glutamine, cysteine and lysine. In some embodiments, connector by the amino acid of multiple non-steric hindrances for exampleGlycine and alanine form. In some embodiments, connector is polyglycine, polyalanine, glycine and alanineCombination (for example poly-(Gly-Ala)) or the combination (for example poly-(Gly-Ser)) of glycine and serine. In other embodimentIn, connector comprises 1 to 20 amino acid, and it is selected from non-natural amino acid. And the connector pair of 3-15 amino acid residueBe preferred in puting together with the part of prolong half-life, the present invention considers the connector of any length or composition. Preferred aminoAcid connector is the O2Oc of following formula:

Or its repetitive.

The connector of describing in literary composition is exemplary, and connector longer and that comprise other residue is also by the present inventionInstitute considers. The present invention also considers non-peptide connector.

The coupling part of connector can comprise one or more alkyl groups, alkoxy base, alkenyl group, cycloalkylGroup, aromatic yl group, heteroaryl groups and heterocyclic group or its combination. For example, may use alkyl to connect style as – NH-(CH₂) s-C (O)-Huo – S-(CH₂) z-C (O)-Huo – O-(CH₂) z-C (O)-, wherein z is 2-20. These alkyl connectors mayFurther included but not limited to low alkyl group (for example C1-C6), lower acyl, halogen (example by the group of any non-steric hindranceAs Cl, Br), CN, NH2 or phenyl replace.

Connector may be also polymer property. Connector can comprise polymerization Biostatic or biodegradableThing chain or unit. Depend on key unstability, there is the polymer that repeats to connect and under physiological condition, can have different steadyDetermine degree. Polymer can contain for example Merlon of key (O-C (O)-O-), polyester (C (O)-O-), polyurethaneEster (NH-C (O)-O-), polyamide (C (O)-NH-). These keys provide with way of example, can be in the present invention but be not intended to limitPolymer chain or connector in the type of the key applied. Suitable polymer comprises for example polyethylene glycol (PEG), polyethylene pyrrolePyrrolidone, polyvinyl alcohol, polyaminoacid, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-Methacrylamide, glucan,Glucan derivative, polypropylene glycol, polyoxy ethylization polyalcohol (polyoxyethylatedpolyol), heparin, heparin sheetBeing total to of section, polysaccharide, cellulose and cellulose derivative, starch and starch derivatives, poly-alkane glycol and its derivative, poly-alkane glycolPolymers and its derivative, polyethylene ethylether etc. and its mixture. Polymer connector is for example PEG. Exemplary non--peptide connectBody is polyethylene glycol connector:

Wherein connector has 100 to 5000kD for example 100 to 500kD molecular weight.

Preferably, for example (O2Oc) unit of one or more amino acid moieties or glycine or silk ammonia are contained in coupling partAcid, C_1-4Alkylidene-C (O)-, C_1-4Alkylidene ,-NH-C_2-6Alkylidene-NH-or-NH-CH₂CH₂-O-CH₂CH₂-NH-diaminoureaUnit or its combination, and coupling part connects 2 reactive groups or functional group.

Preferably, reactive group or functional group are maleimide, sulfydryl or pyridine-2-base disulphanes base.

Peptide or polypeptide and for being connected to the preparation of peptide-connector construction of part of prolong half-life:

Apelin peptide of the present invention and polypeptide and/or peptide-connector construction can pass through synthetic chemistry method or restructuringThe combination results of method or these two kinds of methods. Apelin peptide and/or peptide-connector construction can maybe can be done with total length preparationFor non-full length fragment synthesizes and connects. Peptide of the present invention and polypeptide can be prepared by itself becoming known for the synthetic method of peptide.Can be that any solid phase is synthetic and liquid phase is synthetic for the synthetic method of peptide. Therefore, target peptide and polypeptide can by condensationThe partial peptide of formation albumen or amino acid and its remainder assign to prepare, in the time that product has blocking group, and deprotection group,Can prepare thus the peptide of expectation. Comprise with described in Publication about Document (1)-(5) for the known method of condensation and deprotectionMethod.

(1) M.Bodanszky and M.A.Ondetti, PeptideSynthesis, IntersciencePublishers,NewYork,1966,

(2) Schroeder and Luebke, ThePeptide, AcademicPress, NewYork, 1965,

(3) the people .FundamentalsandExperimentsinPeptide such as NobuoIzumiyaSynthesis,Maruzen,1975,

(4) HaruakiYajima and ShumpeiSakakibara, BiochemicalExperimentSeries1, ProteinChemistryIV, 205,1977, and

(5) HaruakiYajima (editor), DevelopmentofDrugs-Continued, 14, PeptideSynthesis,HirokawaShoten。

After reaction, can for example, by usual purification technique (solvent extraction, column chromatography, liquid chromatogram and recrystallization)Combination comes purifying isolated peptides. In the time that the peptide that as above separated is free cpds, can convert it into by known method suitableShould salt. On the contrary, if the product separating is salt, can convert it into free peptide by known method.

Can be applicable to the amidated acid amides that obtains polypeptide for the synthetic resin of peptide by use. Described resin comprisesChloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxy benzylalcohol resin, 4-methyldiphenyl methylamineResin, PAM resin, 4-hydroxymethyl aminomethyl phenyl acetylamino methyl resin, polyacrylamide resin, 4-(2', 4'-diformazanOxygen base phenyl-methylol) phenoxy resin, 4-(2', 4'-Dimethoxyphenyl-Fmoc-aminomethyl) phenoxy resin, 2-chlorineTrityl chloride resin etc. Use this resinoid, by known various condensation technology itself, make side according to the sequence of target peptideThe alpha-amido of chain and functional group be suitably amino acid condensation on this resin of protection. In the time that serial reaction finishes, from treeFat removes peptide or protected peptide, and removes blocking group, and forms disulfide bond in the situation that of needs, many to obtain targetPeptide.

For the condensation of above-mentioned protected amino acid, can use multiple for example, for the synthetic activating reagent of peptide, HATU, HCTUOr for example carbodiimide. Carbodiimide comprises DCC, N, N'-DIC and N-ethyl-N'-(3-dimethylaminoPropyl group) carbodiimide. For the activation that utilizes this type of reagent, can use racemization inhibitor additive, for example HOBt orOxymaPure. Protected amino acid can be added directly in resin or can together with activating reagent and racemization inhibitorPreactivated is symmetric anhydride, HOBt ester or HOOBt ester, is then added in resin. For protected amino acid whose activationOr can suitably be selected from the known solvent that can be used for peptide condensation reaction with the solvent of resin condensation. For example, that can mention is N, N-Dimethyl formamide, N-methylpyrrole pyridine ketone, chloroform, trifluoroethanol, methyl-sulfoxide, DMF, pyridine, twoAlkane, carrene,Oxolane, acetonitrile, ethyl acetate or their suitable mixture.

Reaction temperature can be selected from and knownly so far can be used for the scope that peptide bond forms, and is generally and is selected from approximately-20 DEG C-50 DEG CScope. The amino acid derivativges of activation conventionally with 1.5-4 doubly excessive ratio use. If by utilizing the test of ninhydrin reactionDiscovery condensation is insufficient, can in the situation that not removing blocking group, repeat condensation reaction to realize abundant condensation. If heavyMultiple condensation still cannot provide the condensation of enough degree, and available acetic anhydride or acetyl imidazole make unreacted glycyl.

Blocking group for the amino acid whose amino of parent material comprises Z, Boc, uncle-pentyloxy carbonyl, isobornyl oxygenBase carbonyl, 4-methoxyl group benzyloxy base carbonyl, CI-Z, Br-Z, adamantyl oxygen base carbonyl, trifluoroacetyl group, phthalyl,Formoxyl, 2-nitrobenzophenone sulfinyl, diphenylphosphothioy (diphenylphosphinothioyl) or Fmoc. Can useCarboxy protective group include but not limited to above-mentioned C_1-6Alkyl, C_3-8Cycloalkyl and C_6-10Aryl-C_1-2Alkyl and 2-Buddha's warrior attendantAlkyl, 4-nitrobenzyl, 4-methoxy-benzyl, 4-chlorobenzyl, phenacyl, benzyloxycarbonyl hydrazide group, uncle-butoxy carbonylBase hydrazide group and trityl hydrazide group.

The hydroxyl of serine and threonine can be protected by esterification or etherificate. The group that is applicable to described esterification comprises carbonDerivative group, such as low-grade alkane acidyl (such as acetyl group etc.), aroyl (such as benzoyl etc.), benzyloxycarbonyl and secondOxygen base carbonyl. The group that is applicable to described etherificate comprises benzyl, THP trtrahydropyranyl and the tert-butyl group. The protection of the phenolic hydroxyl group of tyrosineGroup comprises Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z and the tert-butyl group.

The blocking group of the imidazoles of histidine comprises Tos, 4-methoxyl group-2,3,6-triethylbenzene sulfonyl, DNP, benzyloxyYlmethyl, Bum, Boc, Trt and Fmoc.

The activated carboxyl of initial amino acid comprises corresponding acid anhydrides, azide and active ester, for example with such as pentachlorophenol,2,4,5-trichlorophenol, 2,4,6,-T, 2,4-DNP, Glycolonitrile, p-nitrophenol, HONB, N-hydroxy-succinamide, N-hydroxylThe ester that the alcohol such as base phthalimide, HOBt form. The activation amino of initial amino acid comprises corresponding phosphamide.

The method of eliminating blocking group is included in catalyst (for example palladium black or target carbon) and has the lower hydrogen catalytic reduction that uses,Carry out acid treatment with anhydrous hydrofluoric acid, methanesulfonic acid, TFMS, trifluoroacetic acid or these sour mixtures, use diisopropylEthamine, triethylamine, piperidines, piperazine carry out alkali treatment, in liquefied ammonia, reduce with sodium metal. By above-mentioned acid-treated elimination reactionConventionally at the temperature of-20 DEG C-40 DEG C, carry out, and can advantageously carry out by adding cation receptor, described cation is subject toBody such as methyl phenyl ethers anisole, phenol, thioanisole, m-cresol, p-Cresol, methyl sulfide, Isosorbide-5-Nitrae-succinimide mercaptans, 1,2-dithioglycolDeng. For the protection of 2 of the imidazole group of histidine, 4-dinitrophenyl can be by eliminating with thiophenol processing, and for the protection ofThe formoxyl of the indolyl radical of tryptophan can be by with diluted sodium hydroxide solution or weak aqua ammonia alkali treatment and at 1,2-ethylene dithiolAlcohol, Isosorbide-5-Nitrae-succinimide mercaptans are eliminated by above-mentioned acid treatment under existing.

Method, spendable blocking group, removal for the protection of the functional group that should not participate in initiation material reaction are protectedThe method of protecting the method for group and the functional group of activation wish participation reaction can all have judgement ground from known groups and methodSelect.

Another method of acid amides form that obtains polypeptide comprises: first make C end amino acid-Carboxylamide, thenExtend the peptide chain of N-side until expect chain length, then optionally the alpha-amido of this C-terminal peptide of deprotection and target to be formedThe amino acid of the remainder of polypeptide or the α-carboxyl of peptide, and for example, in mixed solvent (mention above those) condensation instituteState two fragments, wherein the alpha-amido of these two fragments and side chain functional group are protected with suitable blocking group mentioned aboveProtect. The parameter of this condensation reaction can be with mentioned above identical. By said method, from the protected peptide obtaining by condensationThe all blocking groups of middle removal, thus the thick peptide of expectation is provided. Can come purifying this thick peptide freeze-drying by known purifying procedureMain fraction, to provide target amidatioon polypeptide. For obtaining the ester of polypeptide, make the α-carboxyl of C-end amino acid and the alcohol of expectationCondensation, obtains amino-acid ester, and then carries out the program for the manufacture of acid amides mentioned above.

Or recombinant expression method is useful especially. Utilize host cell expression of recombinant proteins (hand control with bagContain the cell of the nucleic acid of encoded peptide sequence, and it will be transcribed and translate and optionally secrete peptide to cell culture medium) be existing skillThe conventional use of art. For recombinant method for production, the nucleic acid of the amino acid sequence of encoded peptide typically synthesizes by conventional method,And be integrated in expression vector. These class methods are particularly preferred for preparation and comprise fusion to other peptide sequence or other albumenThe peptide composition of the peptide of matter or protein fragments or domain. Host cell may optionally be and is at least selected from following one:E.Coli, COS-1, COS-7, HEK293, BHT21, CHO, BSC-1, HepG2,653, SP2/0,293, heLa, myeloma, pouringBar knurl, yeast, insect or plant cell or its any derivative, immobilized or cell of transforming.

Treatment peptide or polypeptide and/or the peptide-connector construction modified comprise reactive group, its can with prolong half-lifePart on the reaction of available reactive functional groups form covalent bond. Reactive group is the chemical based that can form covalent bondGroup. Reactive group can be carboxyl, phosphoryl, carboxyl groups, ester or mixed acid anhydride, maleimide, imino-ester conventionally(imidate), pyridine-2-base-disulphanes base, thereby can with target site at albumin or Fc domain or belowThe functional group of the Fc domain of disclosed chemical modification is as amino group, oh group, carboxylic group or sulfydryl formation covalent bond.For be connected to albuminous interested especially reactive group comprise the group that contains maleimide amino and contain pyridine-The group of 2-base-disulphanes base.

Functional group is the group on albumin or Fc domain, the reactive group on peptide or the polypeptide of modification can with itsReaction forms covalent bond. Functional group comprise for the oh group of the reactive entity bonding of ester; Be used for maleimide, containThere are the group of maleimide amino or the mercapto of pyridine-2-base disulphanes base, imino-ester (imidates) and thioester group reactionBase; Be used for the amino group with carboxylic acid, phosphoryl group, carboxyl groups bonding.

Flow process 1 to 3 is described the synthetic of peptide-connector construction, and wherein peptide is according in formula I to IV described in any onePeptide.

Flow process 1 is described the connector that contains maleimide synthetic of the N-end of the polypeptide that is linked to formula I to IV.

According to the acid amides coupling chemistry of fine foundation, (x is to make the N-end of peptide and one or more O2Oc Amino Acid Unit1 to 20, preferably 1 to 10, and more preferably 3 to 6) coupling produces (1A). (1A) terminal amino functional groups and the acid of activation (1B)(wherein R is alkylidene linear or side chain, aryl, heteroaryl, cycloalkyl or its combination) reaction, produces peptide-contain MalaysiaImido connector construction (1C). The acid (1B) of activation be can business obtain or according to well known by persons skilled in the artTechnology easily obtains from its corresponding carboxylic acid. Preferably, R is linear alkylidene, and more preferably R Shi – CH₂-CH₂-. StandbySelection of land for example, for the peptide (containing the peptide of lysine) that contains amido functional group in side chain, needs orthogonal before coupling reactionProtecting group is Alloc such as, needs subsequently other deprotection steps, to obtain (1C).

Flow process 2A and 2B describe the pyrrole that contains being linked to according to the N-end of the polypeptide described in any one in formula I to IVSynthesizing of the connector of pyridine-2-base-disulphanes base.

Peptide-connector construction (1A) is prepared as described in flow process 1, and further with the acid of the activation of formula (2A)(wherein R ' is alkylidene linear or side chain) reaction, the connector that produces peptide-contain pyridine-2-base-disulphanes base buildsThing (2B). The acid (2A) of activation be can business obtain or according to technology well known by persons skilled in the art from its corresponding carboxylic acidEasily obtain. Preferably, R ’ Shi – CH₂-CH₂-. Alternatively, peptide-connector construction (2C) can adopt HO₂C-R '-SH or itsProtection form (for example trityl or Acm group need other deprotection steps) preparation, further reacts with (2D), producesPeptide-connector construction (2B) of raw peptide-contain pyridine-2-base-disulphanes base.

Adopt diaminourea unit Li as – NH-CH₂CH₂-NH-Huo – NH-CH₂CH₂-O-CH₂CH₂-NH-, with flow process 1,2AWith described in 2B similarly method similar reactive group is linked to the C-end of peptide. This type of peptide-connector constructionLimiting examples is:

Or, can, according to flow process 3A, 3B and 3C, maleimide or pyridine-2-base-disulphanes radical reaction group be connectedKnot is to according to the polypeptide described in any one in formula I to IV:

Adopt standard amide coupling condition, make the C-terminal carboxylic acid group of peptide and one or more O2Oc Amino Acid Unit evenCoproduction raw (3A). End carboxylic acid functional with (3B) or (3C) amino group of (wherein R and R ' are as defined above) reacts, and producesPeptide-connector construction (3D) or (3E) of activation. In addition, for example, in the time that peptide contains carboxyl functional group side chain (Glu or Asp),Need orthogonally protect base (for example O-pi-allyl) and other deprotection steps.

Peptide-connector construction 3F can adopt cysteamine 2-chlorine trityl resin to obtain, then reacts with 3G or 3H,Produce respectively peptide-connector construction 3I or 3E.

Peptide-connector construction (3J) can obtain from diamine resin, and further reacts with (1B) or (2A), produces respectivelyFormula (3K) or peptide-connector construction (3L).

Flow process 1 to 3C is described peptide-connector construction, and it is more used in particular for preparing bioconjugates with albumin. MalaysiaImide reaction group and pyridine-2-base-disulphanes radical reaction group and albuminous cysteine 34 – SH functional groupsReaction.

The preparation of FcAPJ peptide fusion protein

Biogenic poly molecule for example comprises the antibody in the region of containing cysteine that is called at least partly hingeFc can be by recombinant expressed protein product (it is with the secretion of poly (dimerization) form) preparation. The present invention also comprises the Fc of modificationFusion, wherein the amino acid sequence in Fc district is with respect to the amino acid of the Fc-finding in naturally occurring antibody or constant regionSequence changes. For example, Fc-fusion can be used sudden change engineered (modifying), to obtain the FcRn combination of hopeCompatibility/or serum half-life characteristic. The example of the Fc-fusion of modifying has been disclosed in the US patent No. 7,217,798,Be introduced into as a reference.

Fc-fusion of the present invention can also be synthesized change, for example, by connecting connector part and peptide or polypeptidePart changes. In addition having, can be in any expression derived from " modification " Fc-fusion of the Fc domain of recombinant antibodiesSystem comprises preparation or the preparation of employing phage display method in protokaryon and eukaryotic expression system.

Such as Fc-[GGGGS of Fc-connector construction] 2 and Fc-[GGGGS] 3 be described in experimental section below.[GGGGS] 2 and [GGGGS] 3 connectors are linked to the C-end of Fc domain or the N-end of Fc domain.

Bioconjugates

In one embodiment of the invention, puted together and (changed according to the peptide described in any same form in formula I to IV or polypeptide/ covalency links) to the mercapto functional group of albuminous cysteine 34. Aspect of this embodiment, albumin-peptideRefer to that wherein albumin is puted together (chemistry connects) to the bioconjugates of the N-end of peptide. In another embodiment, albumin-Peptide refer to that wherein albumin is puted together (chemistry connects) to the bioconjugates of the C-end of peptide.

In another embodiment of the present invention, merge to people according to peptide or polypeptide described in any same form in formula I to IVOne or more domains in IgG Fc district. The part that antibody comprises two functional independences: be called the variable domain of " Fab ", its knotClose antigen; With the constant domain that is called " Fc ", it relates to for example complement activation of effector function and is attacked by phagocyte. Fc hasLong serum half-life, and Fab is short-lived people such as (, 1989, Nature337:525-31) Capon. When with treatment peptide or manyWhen peptide links together, Fc domain can provide longer half-life (C.Huang, Curr.Opin.Biotechnol.,2009,20,692-699)。

In one embodiment, Fc-peptide refers to bioconjugates, and wherein Fc sequence merges extremely according to appointing in formula I to IVThe N-end of the peptide described in what same form. In another embodiment, peptide-Fc refers to what Fc sequence wherein merged to the C-end of peptideBioconjugates.

Fc district can be that naturally occurring Fc district maybe can be changed to improve some character for example therapeutic properties, circulation timeBetween or reduce assemble.

Passing through and " Fc " domain of antibody merges the useful open No.WO of middle PCT that is modified at of protein for treatment agentIn 00/024782, discuss in detail. The discussion of this file for example, is connected with " carrier " polyethylene glycol (PEG), glucan HuoFc district.

Preferred embodiment of the present invention is to comprise according to any one peptide or polypeptide and prolonging in foregoing embodimentsLong half-lift the bioconjugates of part, wherein the part of prolong half-life be merge to formula I, III, IV by connector orThe Fc domain of the polypeptide of V. In one aspect of the invention, connector has following formula :-[GGGGS] n-, n is 2 or 3, and formulaThe polypeptide of I, II, III or IV contains naturally occurring amino acid. Be suitable for formula I, the II, III or the IV that merge with Fc domainThe example of polypeptide is: QRPRC*SHKGPMC*F, QRPRLSHKC*PMC*F and QRPRLSC*KGPMC*F. One of this embodimentIndividual preferred aspect is the bioconjugates merging as Fc-peptide as defined above, and it comprises as defined modification in literary compositionFor example, in Fc fragment (FcLALA) and formula I to IV peptide or the polypeptide of any same form.

The peptide of having found covalently bound ZhiFc district shows than significantly higher Half-life in vivo of the homologue not merging. ThisOutward, the fusion in YuFc district allows the dimerization/multimerization of polypeptide.

Prepare conjugate:

Flow process 4 and 5 illustrates the peptide described in any same form and prolong half-life in APJ agonist peptide or formula I to IVThe chemical reaction that for example Fc domain of part or albumin are puted together.

Flow process 4 is peptide-connector of Ming Dynasty style 4A and puting together of albuminous cysteine 34 for example.

The wherein coupling part between L representative peptide and maleimide amine functional group. In a special embodiment, L isAs disclosed coupling part in flow process 1,3A, 3B or 3C.

Flow process 5 is peptide-connector construction of Ming Dynasty style 5A and puting together of albuminous cysteine 34 for example.

Wherein L represents Tai with the coupling part between – S-S-pyridine functional groups. In a special embodiment, L is streamDisclosed coupling part in journey 2,3A, 3B or 3C.

The method of the conjugate of describing in preparation flow 1-5 and peptide-connector construction has also been described with example and has been total toWith US provisional application No61/858251 (attorney: PAT055326-US-PSP3) and the 61/858303 (generation of submitting toReason people reel number: PAT055781-US-PSP) in, be introduced into as a reference.

Pharmaceutical composition

Any in can be in many ways of polypeptide of the present invention or its acid amides, ester, salt or its bioconjugates used,Comprise in subcutaneous, intramuscular, intravenous, abdominal cavity, in nose, suction, oral etc. The especially preferred embodiment of the present invention is used thisThe continuous intravenous of bright polypeptide or its acid amides, ester or salt or its bioconjugates is used. Polypeptide of the present invention or biology are puted togetherThing can be used as to be injected or uses within a period of time as continuous infusion. Can use implantable pump. In some reality of the present inventionExecute in scheme, intermittently or continuously polypeptide or bioconjugates are used and are for example continued 1 day, to a couple of days (, 2-3 days or more days) or moreLong-time section, for example, several weeks, several months or several years. In some embodiments, provide intermittence or continuous polypeptide to use at least about 3My god. In other embodiments, provide intermittence or continuous polypeptide or bioconjugates to use at least about 1 week. In other enforcement sideIn case, provide intermittence or continuous polypeptide or bioconjugates to use at least about 2 weeks. During using or use multiple dosage itBetween to maintain average blood plasma peptide concentration may be desired higher than specific threshold. The for example physiological situation based on individual, diseaseSick seriousness etc. is determined expectation concentration. These desired values can be determined by implementation criteria clinical testing. Alternatively, peptide and itsConjugate can be by FcRn mechanism by oral delivery (NatRevImmunol.7 (9), 715-25,2007; NatCommun.3;3:610,2012,AmJPhysiolGastrointestLiverPhysiol304:G262–G270,2013)。

Another aspect, the invention provides and comprise polypeptide of the present invention or and its acid amides, ester, salt or its bioconjugates and oneOr the pharmaceutical composition of multiple pharmaceutically acceptable carrier. This pharmaceutical composition can be formulated for particular route of administration exampleAs, Orally administered, stomach and intestine are used outward and rectal administration etc. In addition, pharmaceutical composition of the present invention can (comprise with solid formBut be not limited to capsule, tablet, pill, particle, pulvis or suppository) or (include but not limited to solution, supensoid agent with liquid formOr emulsion) preparation. Can carry out routine medicine operation (for example sterilizing) and/or can contain conventional inertia dilution pharmaceutical compositionAgent, lubricant or buffer and adjuvant, such as anticorrisive agent, stabilizing agent, wetting agent, emulsifying agent and buffer etc.

The pharmaceutical composition that is suitable for injection generally includes aseptic aqueous solution (in the time of water soluble) or dispersion and for facingTime prepare the aseptic powdery of sterile injectable solution or dispersion.

For intravenous is used, suitable carrier comprise physiological saline, bacteriostatic water, CremophorELTM (BASF,Parsippany, N.J.) or phosphate buffer (PBS). Under all scenario, composition should be all aseptic, and shouldWhen be easy to injection degree flow. Preferred pharmaceutical preparation is stable under manufacture and condition of storage, and necessaryCan for example, the in the situation that of the contamination of opposing microorganism (bacterium and fungi), preserve. Generally speaking, relevant carriers can be to containThere are for example solvent or the decentralized medium of water, ethanol, polyalcohol (for example, glycerine, propane diols and liquid macrogol etc.), and suitableThe mixture closing. Can by for example use such as the dressing of lecithin, the dispersant in the situation that by maintain required particle diameter, withAnd by maintaining adequate liquidity with surface reagent. Can pass through various antibacterial agents and antifungal agent, for example para hydroxybenzeneFormic acid, methaform, phenol, ascorbic acid, thimerosal etc., realize the prevention to microbial action. In many cases, in groupCompound comprises isotonic agent, and for example, sugar, polyalcohol (for example sweet mellow wine), sorbierite, sodium chloride are preferred. Can be by canThe for example aluminum monostearate of material and the gelatin that postpone to absorb are included the absorption that extends Injectable composition in composition in.

Some Injectable composition is isotonic aqueous solution or suspension, and suppository is advantageously from fats emulsion or suspension systemStandby. Described composition can and/or contain adjuvant by sterilizing, and for example anticorrisive agent, stabilizing agent, wetting agent or emulsifying agent, dissolving are urgedEnter salt and/or the buffer of agent, adjusting osmotic pressure. In addition, they can also contain the upper valuable material of other treatment. DescribedPrepared by composition respectively, granulation mixed according to routine or coating method, and containing having an appointment 0.1-75%, or containing having an appointment 1-50%Active component.

Sterile injectable solution can be prepared in the following manner: the reactive compound of aequum is become with cited aboveA kind of or combination in point is mixed in appropriate solvent, as needs, and subsequent filtration sterilizing. Conventionally, dispersion will be by livingProperty compound mixes and contains alkaline decentralized medium and from those sterile carriers of cited required other composition abovePreparation. Under the situation of the aseptic powdery for the preparation of sterile injectable solution, preferred preparation method is vacuum drying and coldFreeze-drying is dry, and it produces active component and from the previous powder through any other desired constituents of its solution of sterilising filtration.

Orally administered composition generally includes inert diluent or edible carrier. The object of using for oral medication, activityCompound can be incorporated in excipient, and with tablet, for example, make containing the form of ingot (troches) or capsule (, gelatine capsule)With. Orally administered composition also can be prepared with fluid carrier with gargling. Pharmaceutically compatible adhesive and/or auxiliary material can be doneFor a part for composition wherein involved. Tablet, pill, capsule, can contain any following composition or have similar containing ingot etc.The compound of character: adhesive, for example microcrystalline cellulose, bassora gum or gelatin; Excipient, for example starch or lactose; Disintegrant,For example alginic acid, Primogel or cornstarch; Lubricant, for example dolomol or Sterotes; Glidant, for example colloid twoSilica; Sweetener, for example sucrose or asccharin; Or flavouring, for example peppermint, gaultherolin or orange essence. Pass for per osThe preparation sending can advantageously be introduced for improvement of the stability in intestines and stomach and/or strengthen the material absorbing.

For using by suction, therapeutic agent of the present invention is preferably from containing suitable propellant (for example,, such as dioxyChange the gas of carbon) pressurizing vessel or distributor or send with aerosol spray form from sprayer. It should be noted that, lung is treatmentThe systemic delivery of agent provides very large surface area.

For example these activating agents can be encapsulated in polymer particle, for example, in the U.S. discloses 20040096403, describeThose, or combine with any in various other medicines delivery vectors known in the art. In other enforcement side of the present inventionIn case, activating agent is combined and is sent with charged lipids, as for example disclosed described in 20040062718 in the U.S.. Should noteArrive, a rear system is for administering therapeutic polypeptide, insulin, and this shows that this system is applicable to administration for peptides material.

Also can be by implementing systemic administration through mucous membrane or through skin mode.

The composition that is applicable to applied dermally comprises the polypeptide of the present invention of effective dose and suitable carrier. Be applicable to through skinThe carrier of sending comprises that help is through acceptable solvent in the absorbability pharmacology of Host Skin. For example, transcutaneous device is for stretching tightBand forms, it comprises backing parts, contain this compound reservoir of (optionally containing carrier), optional speed control barrier(it sends compound to Host Skin with the controlled speed with predetermined in time expand) and device is fixed on skinInstrument.

The composition that is applicable to local application (for example, application to skin and eyes) comprise the aqueous solution, suspension, ointment,Emulsifiable paste, gel or for example sprayable preparation by aerosol delivery etc. This type of local delivery system will be particularly useful for coriumUse. Therefore, they are specially adapted to local application preparation well known in the art, comprise cosmetic formulations. It can contain solubilisingAgent, stabilizing agent, tension force dose, buffer and anticorrisive agent.

As used herein, local application also can relate to suction or intranasal administration. They can use or not use suitable throwingPenetrate in the situation of agent using dry powdered form (separately, for example, as the mixture dry mixture of lactose (with) or blending ingredientsGrain (for example with phosphatide)) from Diskus or with aerosol spray form from pressurizing vessel, pump, injector, atomizer orIn sprayer, send easily.

The present invention further provides pharmaceutical composition and formulation, it comprises, and one or more can reduce as active componentThe material of the compounds of this invention decomposition rate. This type of material that is called " stabilizing agent " herein includes but not limited to that antioxidant is (allAs such as ascorbic acid), pH buffer or salt buffer agent etc.

As used herein, term " pharmaceutically acceptable salt " refers to the biological effectiveness and the property that have kept polypeptide of the present inventionThe salt of matter, and it is conventionally on biology or be not less desirable in other side. Under many situations, polypeptide of the present invention is logicalCross the existence of amino and/or carboxyl or similar group, can form acid and/or alkali salt.

Useful inorganic acid and organic acid form pharmaceutically acceptable acid-addition salts, for example, and acetate, aspartic acidSalt, benzoate, benzene sulfonate, bromide/hydrobromate, bicarbonate/carbonate, disulfate/sulfate, camphorsulfonic acidSalt, chloride/hydrochloride, chlortheophyllonate, citrate, ethanedisulphonate, fumarate, gluceptate,Gluconate, glucuronate, hippurate, hydriodate/iodide, isethionate, lactate, Lactobionate, the moonOsmanthus base sulfate, malate, maleate, malonate, mandelate, mesylate, Methylsulfate, naphthoate, naphthaleneSulfonate, nicotinate, nitrate, octadecane hydrochlorate, oleate, oxalates, palmitate, pamoate, phosphate/phosphor acid hydrogenSalt/dihydric phosphate, Polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate,Toluene fulfonate and trifluoroacetate.

Can be comprised by the inorganic acid of its salt derivative, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc.

Can be comprised by the organic acid of its salt derivative, for example, acetic acid, propionic acid, glycolic, oxalic acid, maleic acid, malonic acid, amberAmber acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, Loprazolam, ethane sulfonic acid, toluenesulfonic acid, sulfosalicylic acidDeng. Can form pharmaceutically acceptable base addition salts with inorganic base and organic base.

Can be comprised by the inorganic base of its salt derivative, for example, the metal of ammonium salt and XII family of periodic table I family to the. At someIn embodiment, described salt is derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc and copper; Especially suitable salt comprise ammonium salt, sylvite,Sodium salt, calcium salt and magnesium salts.

Can be comprised by the organic base of its salt derivative, for example, primary, secondary and tertiary amine, replacement amine comprise naturally occurring replacementAmine, cyclic amine, deacidite etc. Some organic amine comprise isopropylamine, benzyl star (benzathine), choline salt,Diethanol amine, diethylamine, lysine, meglumine, piperazine and tromethamine.

Pharmaceutically acceptable salt of the present invention can be by usual chemical method from parent compound, alkalescence or acidic moietySynthesize. Conventionally, these salt can by make the free acid form of these compounds and stoichiometric suitable alkali (for example Na, Ca,The hydroxide of Mg or K, carbonate, bicarbonate etc.) react to prepare, or by making the free alkali shape of these compoundsFormula reacts to prepare with stoichiometric suitable acid. These react normally in water or organic solvent or the mixing of the twoIn thing, carry out. Conventionally,, feasible in the situation that, use non-aqueous media, as ether, ethyl acetate, ethanol, isopropyl alcohol or acetonitrile areDesirable. The list of other suitable salt can be for example at " Remington'sPharmaceuticalSciences ", and20 editions, MackPublishingCompany, Easton, Pa., (1985); And " the Handbook of Stahl and WermuthofPharmaceuticalSalts:Properties,Selection,andUse”(Wiley-VCH,Weinheim,Germany, 2002) in, find.

As used herein, term " pharmaceutically acceptable carrier " comprises any and all solvents, decentralized medium, dressingMaterial, surfactant, antioxidant, anticorrisive agent (for example antiseptic, antifungal agent), isotonic agent, absorption delay agent, salt, anti-Rotten agent, medicine, medicine stabilizing agent, adhesive, excipient, disintegrant, lubricant, sweetener, flavouring, dyestuff etc. and group thereofClose, it is known (for example, referring to Remington'sPharmaceutical to those skilled in the artSciences, the 18th edition, MackPrintingCompany, 1990, pp.1289-1329). Except itself and not phase of active componentBeyond appearance, in treatment or pharmaceutical composition, consider to use any conventional carrier.

The inventive method:

Apelin peptide family is the native ligand family of unique known G albumen coupling apj receptor. Apelin gene code77 amino acid whose polypeptide, this polypeptide is processed to the biologically active form of apelin peptide, for example apelin-36, apelin-17, the form (Pyr modifying through pyroglutamic acid of apelin-16, apelin-13, apelin-12 and apelin-13¹-Apelin-13). Any in these apelin peptides is being bonded to after apj receptor, by Gi and Gq protein transduction signal. ?In cardiac muscle cell, Gi or Gq coupling cause the variation that internal pH, PLC activation and IP3 produce, thereby strengthen myofilament calcium sensitivityAnd finally increase myocardial contractive power. Gs, adenylyl cyclase and cAMP that Gi coupling suppresses activation produce, and increase pAkt waterFlat, realize Cardioprotective. In vascular endothelial cell, increase nitric oxide (NO) by the APJ activation of Gi, pAKT and produce, itsImprove smooth muscle relaxation, make overall vasodilation.

The patient who suffers from chronic stable heart failure has Decompensated accidental acute attack, wherein myocardial contractive powerFurther decline and severity of symptoms. These increase the weight of to be called acute decompensation DHF (ADHF). Be used for the current of ADHFTherapy comprises diuretics, vasodilator and directly increases the cardiotonic of myocardial contractive power. Current intravenous cardiotonic (DOPA phenolButylamine, dopamine, milrinone, Levosimendan) adverse events, the long-term mortality of for example cardiac arrhythmia and increase, has been peopleInstitute know. Synthetic apelin polypeptide analog of the present invention is provided for the therapy of ADHF, and it increases myocardial contractive power, and does not haveCause cardiac arrhythmia or mortality tendency, and solved the huge unsatisfied medical science needs of chronic heart failure.

In fact, acute human apelin treats (5min), causes the cardiac output of coronary vasodilator diastole and improvement. SoAnd natural apelin shows extremely short t in vivo_1/2(several seconds) and acting duration (a few minutes). With natural apelin phaseRatio, efficiently synthetic apelin peptide agonists of the present invention has the longer half-life.

In cardiac muscle cell, apj receptor activation a) improves myocardial contractive power by Gi/Gq, PLC and Ca2+, and b) passes throughGi, pAkt activation provides Cardioprotective, but can not increase cAMP (being seen as used other cardiotonic). In addition, at endotheliumIn cell, APJ excitement causes arteries diastole, thereby is further of value to heart failure because of the work load of unloading left ventricleExhaust. In a word, these synthetic apelin polypeptide analogs can improve overall cardiac function, reduce cardiac arrhythmia and occur and survival is providedBenefit.

Recently, had multiple preclinical study publications, it pays close attention to Apelin may relate to diabetes and insulinResistance. Show Apelin1) thereby reduce blood sugar level, 2 by the glucose absorption in improvement muscle, fat and heart)Protection pancreatic β cell be not subject to ER stress and follow-up apoptosis, 3) reduce the insulin secretion in β cell, and 4) regulate adipose tissueThe steatolysis (lypolysis) of middle catecholamine induction. The activation of pAKT path has involved these processes.

Pharmaceutically can according to the polypeptide of arbitrary formula in formula I to IV or its of free form or pharmaceutically acceptable salt formSalt or its bioconjugates accepted demonstrate valuable pharmacological property, the exciting character of for example apj receptor, for example as underExternal and the body build-in test providing in face portion is shown, and is therefore applicable to treatment.

Polypeptide of the present invention or its pharmaceutically acceptable salt can be used for treatment and are selected from following indication: acute compensatory mistakeTonality heart failure (ADHF), chronic heart failure, pulmonary hypertension, auricular fibrillation, Bu Lugeda Cotard, chamber property are aroused in interestOverrun, atherosclerotic, hypertension, ISR, ischemic angiocardiopathy, cardiomyopathy, cardiac fibrosis, the rhythm of the heart notTogether, water retention, diabetes (comprising gestational diabetes mellitus), obesity, peripheral arterial disease, cerebrovas-cularaccident, temporary ischemic episode,Traumatic brain injury, ALS, burn (comprising sunburn) and pre-eclampsia.

Therefore, as other embodiments, the invention provides the polypeptide of arbitrary formula in formula I to IV or its acid amides, ester, salt orThe purposes of its bioconjugates, it is used for the treatment of and the active relevant disease of apj receptor. In another embodiment, treatment choosingFrom the disease of the excitement response to apj receptor. In another embodiment, disease is selected from above-mentioned list, is acute generation aptlyRepay dysfunctional heart failure. In the another subset of this embodiment, the invention provides the polypeptide of arbitrary formula in formula I to IV or itsAcid amides, ester, salt or its bioconjugates purposes in the medicine for the preparation of the treatment disease relevant to apj receptor activity.

Therefore, as another embodiment, the invention provides the polypeptide of arbitrary formula in formula I to IV or its acid amides, ester, salt orThe purposes of its bioconjugates in treatment. In another embodiment, this treatment is selected from and can (swashs by the activation of apj receptorMoving) disease for the treatment of.

In another embodiment, the invention provides the method for the disease of the excitement response for the treatment of to apj receptor, its bagDraw together on administering therapeutic can the formula I to IV of receiving amount in polypeptide or its acid amides, ester or the salt of arbitrary formula. In another embodiment,This disease is selected from above-mentioned list, is acute decompensation DHF aptly.

In the another subset of this embodiment, the invention provides the side of the disease that treatment is relevant to the activity of apj receptorMethod, it polypeptide or its acid amides, ester, salt or its biology that comprises arbitrary formula in the formula I to IV of acceptable amount on administering therapeutic is sewedCompound.

The pharmaceutical composition of the present invention using in treatment or the effective dose of combination will depend on, for example, treat content and orderMark. It will be understood by a person skilled in the art that, therefore the suitable dosage level being used for the treatment of incites somebody to action partly according to changing below: institute passsThe molecule sending, size (body weight, body surface or the organ chi that uses indication, route of administration and the patient of fusion variantVery little) and situation (age and health status). Therefore, clinician can determine dosage and change route of administration to obtain optimal treatmentEffect. According to above-mentioned factor, exemplary dosage can be the paramount scope to about 100mg/kg of approximately 0.1 μ g/kg, or higher. At itIn its embodiment, dosage range can be that 0.1 μ g/kg is until about 100mg/kg; Or 1 μ g/kg until about 100mg/kg.

Administration frequency will depend on the pharmacokinetic parameter of the dual-use function albumen in preparation used. Conventionally clinical doctor,Teacher will use composition until reach the dosage that can realize desired effects. Therefore, composition can be used as single dose and uses, on timeBetween use (the expectation molecule that each dosage can or can not contain same amount) with two or more dosage, or pass through implanted deviceOr conduit is used in continuous infusion mode. Suitably the further segmentation of dosage is that those skilled in the art implement also in a usual mannerIn the scope of the normal work to do of implementing at them. Can be by determining suitable dosage by suitable dose-response data.

" the treatment effective dose " of term polypeptide of the present invention refers to and excites individual biology or medical response (for example, to improve diseaseShape, alleviate the patient's condition, slow down or postpone disease process or prevent disease etc.) the amount of polypeptide of the present invention. A non-limiting realityExecute in scheme, term " treatment effective dose " refers in the time being applied to individuality, and effectively (1) alleviates at least in part, suppresses, preventsAnd/or improve that (i) improve by the activation of apj receptor or (ii) relevant to the activity of apj receptor or (iii) with APJThe abnormal activity of acceptor is the patient's condition, illness or disease or its symptom of feature; Or the polypeptide of the present invention of (2) activation apj receptorAmount.

In another non-limiting embodiments, term " treatment effective dose " refers to when being applied to cell or tissue or non-When cell biological material or medium, the amount of the polypeptide of the present invention of at least part of activatable apj receptor effectively. Art technologyPersonnel should be appreciated that, the absolute effective dose of concrete activating agent can be according to changing such as following factor: the biology terminal of expectation,Activating agent, the target tissue etc. sent. Those skilled in the art understand, and " treatment effective dose " can be used with single dose, orCan realize by using multiple dosage. For example, in the case for the treatment of activating agent in heart failure, effective dose can be to be enough toRealize the amount of patient's clinical improvements, for example, the fluid retention of the motion tolerance/ability of increase, the blood pressure of increase, minimizing and/Or the result of the cardiac function quantitative experiment improving, for example, LVEF, locomitivity (to the time that power exhausts) etc.

As used herein, term " individuality " refers to animal. Conventionally, animal is mammal. Individuality also refers to, for example, clever longClass (for example, the mankind), ox, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird etc. In certain embodiments, individualBody formula primate. In other embodiments, individuality is the mankind.

As used herein, term " inhibition " (" inhibit ", " inhibition " or " inhibiting ") refer to alleviate orConstrain the given patient's condition, symptom or illness or disease, or significantly reduce the baseline activity of biologically active or process.

In one embodiment, as used herein, term " treatment " (" treat ", " treating " or " treatment ")Arbitrarily disease or illness refer to that improving this disease or illness (, slows down or stop or alleviate this disease or its at least one is clinicalThe development of symptom). In another embodiment, " treatment " refers to and alleviates or improve at least one body parameter, comprise that patient is notThose that can experience. In yet another embodiment, " treatment " refer to aspect health regulation and control disease or illness (for example, stableThe symptom that can experience) or aspect physiology regulation and control disease or illness (for example, stablizing body parameter) or the two. One real againExecute in scheme, " treatment " refers to prevention or postpones outbreak or development or the deterioration of disease or illness.

As used herein, term " prevention " (" prevent ", " preventing " and " prevention ") refers to preventionRecurrence, outbreak or the development of one or more symptom of body illness, this is for example, using or treatment group by treatment (therapeutic agent)Closing using of (for example, the combination of therapeutic agent) produces.

As used herein, if individuality will be on biology, medically or benefit from treatment in quality of life, thisThis treatment of body " needs ".

As used herein, unless otherwise indicated herein or the obvious contradiction of context, otherwise in the context of the invention (especiallyIn claim context) in term "/kind " (" a ", " an ") used, " being somebody's turn to do " (" the ") and similar terms understandFor contained odd number with plural number the two.

Unless otherwise indicated herein or in addition obviously contradiction of context, otherwise all methods described herein all can be fitted arbitrarilyShould order implement. Use any and all examples or exemplary language (for example, as " ") provided herein is only intended to explain betterBright the present invention, and claimed scope of the present invention is not carried out to any restriction.

The activity of polypeptide of the present invention can be evaluated by in-vitro method hereinafter described.

The component analysis of hAPJ calcium current:

Chem-5APJ stabilized cell (Millipore#HTS068C) is flat with 10,000 cells/well with 384 hole patternsBe laid in 25ul growth medium, then in 37 DEG C of incubator for tissue cultures, grow 24 hours. In analysis first 1 hour, add 25ul/Hole FLIPR calcium 4 dyestuffs (MolecularDevicesR8142) and 2.5mM probenecid, and cultivate in 37 DEG C of incubator for tissue culturesCell 1 hour. Peptide is dissolved in HBSS, HEPES&0.1%BSA buffer solution, and 10 times of triplicate serial dilutions, from 50uMTo 5pM. Use FLIPRTetra peptide to be added into (1:5, final peptide concentration is 10uM to 1pM) in the cell with dyestuff. CarefullyThe FLIPR dyestuff of born of the same parents inside is being bonded to emitting fluorescence after calcium, from the crested of fluorescence of outside. At FLIPRTetra is upper measures fluorescence with 470-495 excitation wavelength and 515-575 emission wavelength. Add and start to carry out for first 10 seconds at peptideReading, amounts to reading 3 minutes. Calculate maximum-minimum of a value mapping for each peptide concentration, and the stimulation to calcium current amount for peptideCarry out the EC at calculated curve flex point place with GraphPadprism software₅₀Value.

Plasma stability is analyzed:

Material:

Working solution: prepare 1mg/mL test article in Milli-Q water

Extract solution_：There is the methyl alcohol of 0.1% formic acid and 400ng/mL glibenclamide (Glyburide): acetonitrile: water (1:1:1)。

Blood plasma: male Sprague-Dawley rat blood plasma (thering is liquaemin), purchased from BioreclamationLLC(Liverpool,NY)

Whole blood: male SpragueDawley whole blood (thering is liquaemin), purchased from BioreclamationLLC(Liverpool,NY)

LH: male rat SpragueDawley lung is purchased from BioreclamationLLC (Liverpool, NY).Adding after 5x volume 1XPBS, using polytron homogenizer that lung is homogenized. At 4 DEG C, homogenate is centrifugal with 9000rpm10min. By supernatant with 3000rpm centrifugal 30min again, to prepare transparent supernatant. Use commercial reagent box (Pierce,ThermoScientific) measure protein concentration.

Sample preparation program: (peptide)

In following bio-matrix, prepare test substances: heparinize rat plasma, heparinize rat whole blood or LH. LogicalCross the working solution of 5uL1mg/mL is added in 995uL rat plasma or whole blood, prepare blood plasma and whole blood with 5000ng/mLSample. Prepare in the following manner LH sample: LH is diluted to 1mg/ml albumen with phosphate buffered saline (PBS) (PBS)Concentration, is added into 5uL working solution in the LH of 995uL dilution subsequently. In water bath incubator, at 37 DEG C, cultivate sampleProduct slight concussion (65～75rpm). At 0min, 5min, 15min, 30min, 60min, 120 and when 240min, will cultivate sampleThe 25uL aliquot of product is transferred in 96 orifice plates and exists side by side and carry out protein precipitation with 150uL extraction solution. Test in cultivationAfter, at 4 DEG C by sample panel with 4000rpm centrifugal 10 minutes. After this, use liquid-transfering device (TecanTemo) that supernatant is turnedMove in another plate, and add 50uL water to all samples. Before analyzing, LC-MS makes the vibration of plate vortex.

Sample preparation program (conjugate)

By 5uL1mg/mL working solution is added in 495uL rat plasma, prepare test article with 50,000ng/mL.Sample is cultivated under 37 DEG C, gentle jolting (65～75rpm) in water bath incubator. Time 0hr, 0.5hr, 1hr, 2hr,4hr, 6 and 24hr, cultivates 50uL the aliquot of sample and transfers to 96 orifice plates, by 100uL40mMTCEP (three (2-carboxylsEthyl) phosphine) add to each sample. Reactant mixture is cultivated 1 hour at 37 DEG C. After having reacted, adopt 300uL acetonitrile precipitation eggWhite matter. By sample panel 4 DEG C with 4000rpm centrifugal 10 minutes. Then, use liquid-transfering device (TecanTemo) to shift 125uLSupernatant, to another plate, adds 50uL water to all samples. Before LC-MS analyzes, by plate vortex.

The LC-MS of sample stability analyzes

HPLC: the Agilent1290HPLC with automatic sampler

Post: MAC-MODACEC18,3 μ m, 30mmx2.1mmi.d.

Mobile phase A: 0.1% formic acid in acetonitrile

0.1% formic acid of Mobile phase B: Yu Shuizhong

Gradient program

Mass spectrum: AgilentQ-TOF6530

Data acquisition scheme: the full scan that uses 100 – 1000m/z mass ranges

Data acquisition and analysis software: MassHunter

Data analysis:

Stability analysis: by changing into remaining with respect to initial (t=0) peak area at the peak area of each time pointRemaining percentage, measures stability half-life (t1/2) value.

Residue percentage=100x (sample peak area) ÷ (t=0 peak area)

Calculate the natural logrithm of residue percent value and for sample time mapping (MicrosoftExcel). Pass through lineProperty returns to measure the slope k (MicrosoftExcel) of this straight line.

Then carry out the computational stability half-life by formula t1/2=0.693 ÷ k.

Plasma stability analysis based on substitute activity:

Carry out calcium current amount scheme mentioned above, and carry out following variation. Also use 5% rat plasma(Bioreclamation#RATPLNAHP-M processes through heparin Na) cultivates peptide. In 37 DEG C of incubator for tissue cultures cultivate after, timeBetween put t₀And t₂₄Hour carry out reading. Estimate peptide plasma half-life (taking minute as unit) to get off by calculating:

1)LN((t₀Time EC₅₀)/(t_{24 hours}Time EC₅₀)),

2) calculate the above slope being worth, and

3)t_1/2=0.693/ (slope ^2)

Adopt test analysis (as described above), polypeptide of the present invention demonstrate the table 2 that below provided and 3 effect andStability.

Table 2: the activity of polypeptide and stability

Table 3: the relation between plasma stability analysis and the analysis of the plasma stability based on substitute activity:

Polypeptide of the present invention or its bioconjugates can have and apelin-13 or the similar APJ of pyr-1-apelin-13Acceptor effect. In one embodiment, polypeptide of the present invention has the EC that is less than 100nM₅₀. In another embodiment, originallyThe polypeptide of invention or its bioconjugates have and are less than 50nM, are preferably less than 25nM and are more preferably less than the EC of 15nM₅₀. Again oneIn embodiment, polypeptide of the present invention or its bioconjugates have the EC that is less than 10nM₅₀。

Polypeptide of the present invention or its bioconjugates can have the blood plasma that is better than apelin-13 or pyr-1-apelin-13Stability. In one embodiment, plasma stability improves at least 2 times. In one embodiment, polypeptide of the present invention orIts bioconjugates has the plasma stability of at least 30 minutes. In another embodiment, polypeptide of the present invention or its biologyConjugate has at least 60 minutes, at least 80 minutes, preferably at least 100 minutes and more preferably the blood plasma of at least 150 minutes is steadyQualitative.

Polypeptide of the present invention or its bioconjugates can be in using one or more other therapeutic agents or before orUse afterwards. Polypeptide of the present invention or bioconjugates can be separated and execute with other activating agent by identical or different route of administrationWith, or use together with other activating agent in same drug composition.

In one embodiment, the invention provides product, the polypeptide of arbitrary formula or its acyl in its contained I to IVAmine, ester, salt or its bioconjugates, and at least one other therapeutic agent, its as in treatment simultaneously, separately or sequentially makeWith combination preparation. In one embodiment, this treatment is the disease of the activation response to apj receptor or the treatment of the patient's condition.

The product providing as combination preparation comprises composition, and described composition wraps together in same pharmaceutical compositionContaining polypeptide or its acid amides, ester, salt or its bioconjugates and other therapeutic agent of arbitrary formula in formula I to IV, or to separateIn the contained I to IV of form (for example, with kit form) polypeptide of arbitrary formula or its acid amides, ester, salt or its bioconjugates andOther therapeutic agent.

In one embodiment, the invention provides pharmaceutical composition, in its contained I to IV the polypeptide of arbitrary formula orIts acid amides, ester, salt or its bioconjugates and other therapeutic agent. Optionally, this pharmaceutical composition can comprise as described abovePharmaceutically acceptable excipient.

In one embodiment, the invention provides the medicine box that comprises two or more pharmaceutical compositions that separate, instituteState the polypeptide or its acid amides, ester, salt or its bioconjugates that one of at least contain arbitrary formula in formula I to IV of pharmaceutical composition.In one embodiment, this medicine box comprises the device for keeping independently these compositions, for example container, discrete bottle or pointVertical paillon foil bag. The example of this type of medicine box is as being generally used for the blister package of package troche, capsule etc.

Medicine box of the present invention can be used for using different dosage form (for example, oral and stomach and intestine are outer), uses with different spacing of dosesIndependent groups compound, or for adjusting each other the dosage of independent groups compound. For contributing to compliance, medicine box of the present invention wraps conventionallyContaining the description for using.

In combined therapy of the present invention, peptide of the present invention or polypeptide or bioconjugates and other therapeutic agent can be by identicalOr different manufacturers manufacture and/or preparation. In addition, can by peptide of the present invention or bioconjugates and other therapeutic agent as follows together withBring in combined therapy: (i) before granting combination product is to doctor, (for example comprising the compounds of this invention and other therapeutic agentThe situation of medicine box under); (ii) before being about to use by doctor self (or under doctor instructs); (iii) by patient oneself, exampleAs during sequentially using polypeptide of the present invention or bioconjugates and other therapeutic agent.

Therefore, the invention provides the polypeptide of arbitrary formula in formula I to IV or the use of its acid amides, ester, salt or its bioconjugatesOn the way, it is used for the treatment of disease or the patient's condition of the excitement response to apj receptor, wherein this medicine be produced with another therapeutic agent oneRise and use. The present invention also provides the purposes of another therapeutic agent, and it is used for the treatment of the disease of the excitement response to apelin acceptorOr the patient's condition, wherein the polypeptide of this medicine and arbitrary formula in formula I to IV or its acid amides, ester, salt or its bioconjugates together with executeWith.

The present invention also provides polypeptide or its pharmaceutically acceptable salt or its bioconjugates of arbitrary formula in formula I to IV,It is used for the treatment of in the disease of excitement response or the method for the patient's condition to apj receptor, the wherein polypeptide of arbitrary formula in this formula I to IVOr its acid amides, ester, salt or its bioconjugates are produced for using with together with other therapeutic agent. It is another that the present invention also providesOuter therapeutic agent, it is used for the treatment of in the disease of excitement response or the method for the patient's condition to apj receptor, wherein this other treatmentAgent is produced for using together with the polypeptide of the arbitrary formula of formula I to IV or its acid amides, ester, salt or its bioconjugates. ThisBright polypeptide or its acid amides, ester, salt or its bioconjugates that arbitrary formula in formula I to IV is also provided, it is used for the treatment of APJ is subject toIn the disease of the excitement response of body or the method for the patient's condition, the polypeptide of arbitrary formula or its acid amides in its Chinese style I to IV, ester, salt or itsBioconjugates is used with together with other therapeutic agent. The present invention also provides other therapeutic agent, and it is used for the treatment of APJ is subject toIn the disease of the excitement response of body or the method for the patient's condition, wherein in this other therapeutic agent and formula I to IV the polypeptide of arbitrary formula orIts acid amides, ester, salt or its bioconjugates are used together.

The present invention also provides the polypeptide of arbitrary formula or the use of its acid amides, ester, salt or its bioconjugates in formula I to IVOn the way, it is used for the treatment of disease or the patient's condition of the excitement response to AJP acceptor, and wherein this patient previous (for example, in 24 hours)Via other therapeutic agent treatment. The present invention also provides the purposes of other therapeutic agent, and it is used for the treatment of the excitement to apj receptorDisease or the patient's condition of response, wherein this patient previously (for example, in 24 hours) by the polypeptide of arbitrary formula in formula I to IV orIts acid amides, ester, salt or the treatment of its bioconjugates.

In one embodiment, other therapeutic agent is selected from cardiotonic, Beta-3 adrenergic receptor blocking agent, HMG-Co-AReductase inhibitor, angiotensin II receptor antagonists, ACE (ACE) inhibitor, calcium channel blocker(CCB), endothelin antagonist, renin inhibitor, diuretics, ApoA-I analogies, antidiabetic, anoretic, aldosterone are subject toBody blocking agent, blockade of endothelin receptors agent, aldosterone synthase inhibitors (ASI), CETP inhibitor, anti-coagulants, relaxain, BNP(Nesiritide) and nep inhibitor.

Term and the second activating agent or treatment " combination " comprise uses polypeptide of the present invention altogether (for example,, according in formula I to IVThe polypeptide of arbitrary formula or polypeptide or the bioconjugates otherwise described herein) and the second activating agent or treatment; First executeWith the compounds of this invention, use subsequently the second activating agent or treatment; And first use the second activating agent or treatment, use subsequentlyCompound of the present invention.

The disease that term " the second activating agent " comprises treatment known in the art, prevents or alleviate disease described herein or illnessAny activating agent of shape, described disease or illness are for example illness or the disease of the activation response to apj receptor, for example, acuteDecompensation DHF (ADHF), chronic heart failure, pulmonary hypertension, auricular fibrillation, Bu Lugeda Cotard, chamberProperty tachycardia, atherosclerotic, hypertension, ISR, ischemic angiocardiopathy, cardiomyopathy, cardiac fibrosis,Cardiac arrhythmia, water retention, diabetes (comprising gestational diabetes mellitus), obesity, peripheral arterial disease, cerebrovas-cularaccident, temporary lackingBlood outbreak, traumatic brain injury, ALS, burn (comprising sunburn) and pre-eclampsia.

The example of the second activating agent comprises that cardiotonic, Beta-3 adrenergic receptor blocking agent, HMG-Co-A reductase suppressAgent, angiotensin II receptor antagonists, ACE (ACE) inhibitor, calcium channel blocker (CCB), endotheliumElement antagonist, renin inhibitor, diuretics, ApoA-I analogies, antidiabetic, anoretic, aldosterone receptor blocking agent, inSkin element receptor blocking pharmacon, aldosterone synthase inhibitors (ASI), CETP inhibitor, anti-coagulants, relaxain, BNP (Nesiritide)And/or nep inhibitor.

As used herein, cardiotonic comprises for example dobutamine, isoproterenol, milrinone, Amrinone(amirinone), Levosimendan, adrenaline (epinephrine), norepinephrine (norepinephrine), isopropylBase adrenaline and digoxin.

As used herein, Beta-3 adrenergic receptor blocking agent comprises for example acebutolol (acebutolol), atenolol(atenolol), betaxolol (betaxolol), bisoprolol (bisoprolol), carteolol (carteolol), Mei TuoLuo Er (metoprolol), Nadolol (nadolol), Propranolol (propranolol), Sotalol (sotalol) andTimolol (timolol).

As used herein, anti-coagulants comprises DALT (Dalteparin), danaparoid (Danaparoid), Enoxaparin(Enoxaparin), heparin, TINZ (Tinzaparin), warfarin (Warfarin).

Term " HMG-Co-A reductase inhibitor " (presses down also referred to as beta-hydroxy-Beta-methyl glutaryl-CoA-reductasePreparation) comprise and can be used for reducing the activating agent of (comprising cholesterol) of lipid level in blood. Example comprises Atorvastatin(atorvastatin), cerivastatin (cerivastatin), mevastatin (compactin), dalvastatin(dalvastatin), dihydro mevastatin (dihydrocompactin), fluorine statin (fluindostatin), Fluvastatin(fluvastatin), Lovastatin (lovastatin), Pitavastatin (pitavastatin), mevastatin(mevastatin), Pravastatin (pravastatin), Rosuvastatin (rosuvastatin), auspicious his spit of fland of cutting down(rivastatin), Simvastatin (simvastatin) and Wei Luotating (velostatin) or its pharmaceutically acceptable salt.

Term " ACE-inhibitor " (also referred to as ACEI) comprises that interruption angiotensin I is to bloodThe molecule of the enzyme degraded of angiotensin II. This compounds can be used for regulating blood pressure and treatment congestive heart failure. Example bagDraw together alacepril (alacepril), benazepil (benazepril), Benazeprilat (benazeprilat), captopril(captopril), ceronapril (ceronapril), Cilazapril (cilazapril), Delapril (delapril), according to thatPuli (enalapril), enalapril draw (enaprilat), fosinopril (fosinopril), Imidapril(imidapril), lisinopril (lisinopril), Moexipril (moexipril), Moveltipril (moveltopril), trainingDiindyl Puli (perindopril), quinapril (quinapril), Ramipril (ramipril), Spirapril (spirapril),Temocapril (temocapril) and Trandolapril (trandolapril) or its pharmaceutically acceptable salt.

Term " endothelin antagonist " comprises Bosentan (bosentan) (referring to EP526708A), tezosentan(tezosentan) (referring to WO96/19459) or its pharmaceutically acceptable salt.

Term " renin inhibitor " comprises ditekiren (ditekiren) (chemical name: [1S-[1R*, 2R*, 4R*(1R*, 2R*)]]-1-[(1,1-dimethyl ethyoxyl) carbonyl]-L-prolyl-L-phenyl alanyl-N-[2-hydroxyl-5-Methyl isophthalic acid-(2-methyl-propyl)-4-[[[2-methyl isophthalic acid-[[(2-pyridylmethyl) amino] carbonyl] butyl] amino] carbonyl] oneselfBase]-N-Alpha-Methyl-L-Histidine acid amides); Terlakiren (terlakiren) (chemical name: [R-(R*, S*)]-N-(4-morpholineBase carbonyl)-L-phenyl alanyl-N-[1-(cyclohexyl methyl)-2-hydroxyl-3-(1-methyl ethoxy)-3-oxopropyl]-S-methyl-Cys acid amides); Aliskiren (Aliskiren) (chemical name: (2S, 4S, 5S, 7S)-5-amino-N-(2-Carbamyl-2,2-dimethyl ethyl)-4-hydroxyl-7-{[4-methoxyl group-3-(3-methoxy propoxy) phenyl] methyl }-8-Methyl-2-(third-2-yl) pelargonamide) and zankiren (Zankiren) (chemical name: [1S-[1R*[R* (R*)], 2S*, 3R*]]-N-[1-(cyclohexyl methyl)-2,3-dihydroxy-5-methyl hexyl]-α-[[2-[[(4-methyl isophthalic acid-piperazinyl) sulphonyl] methyl]-1-oxo-3-phenyl propyl]-amino]-4-thiazole propionamide) or its hydrochloride or the SPP630 being developed by Speedel,SPP635 and SPP800, or formula (A) and RO66-1132 (B) and RO66-1168:

OrIts pharmaceutically acceptable salt.

If not clearly definition, term " aliskiren " is interpreted as free alkali and salt thereof, and especially it pharmaceutically canThe salt of accepting, most preferably its hemifumarate.

Term " calcium channel blocker (CCB) " comprises dihydropyridines (DHPs) and non-DHPs (for example, diltiazem(diltiazem) type and Verapamil (verapamil) type CCB). Example comprises Amlodipine (amlodipine), the general ground of benzylThat (Bepridil), diltiazem, felodipine (felodipine), Rui Xiding (ryosidine), isradipine(isradipine), lacidipine (lacidipine), nicardipine (nicardipine), nifedipine (nifedipine),Niguldipine (niguldipine), niludipine (niludipine), Nimodipine (nimodipine), Nisoldipine(nisoldipine), nitrendipine (nitrendipine), Verapamil and Nilvadipine (nivaldipine), preferablyBe selected from following non--DHP representative: flunarizine (flunarizine), prenylamine (prenylamine), diltiazem,Fendiline (fendiline), gallopamil (gallopamil), mibefradil (mibefradil), Anipamil(anipamil), tiapamil (tiapamil) and Verapamil or its pharmaceutically acceptable salt. CCB can be used as anti-hypertensionMedicine, antianginal drug or antiarrhythmics.

Term " diuretics " comprises thiazine derivative (for example, chlorothiazide, Hydrochioro, methychlothiazide and chlorthalidone).

Term " ApoA-I analogies " comprises D4F peptide (for example, formula D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F)。

Angiotensin II receptor antagonists or its pharmaceutically acceptable salt are interpreted as can be in conjunction with Angiotensin IIThe AT of acceptor₁-receptor subtype but can not activate the active component of this receptor. As suppressing AT₁The result of-acceptor, these antagonistsCan for example be used as antihypertensive or be used for the treatment of congestive heart failure.

This class AT₁Receptor antagonist comprises the compound with different structure feature, and especially preferred is non-peptide class. ExampleAs, can mention and be selected from following compound: Valsartan (valsartan), Losartan (losartan), Candesartan(candesartan), Eprosartan (eprosartan), Irbesartan (irbesartan), saprisartan(saprisartan), Tasosartan (tasosartan), Telmisartan (telmisartan), there is the called after E-of following formula1477 compound

There is the compound of the called after SC-52458 of following formula

And there is the compound of the called after ZD-8731 of following formula

Or in each case, its pharmaceutically acceptable salt.

Preferred AT₁-receptor antagonist is Candesartan, Eprosartan, Irbesartan, Losartan, Telmisartan, figured silk fabricsSha Tan. Further preferably those commercially available activating agents, most preferably are Valsartan or its pharmaceutically acceptable salt.

Term " antidiabetic " comprises can promote the insulin secretion enhancers of insulin from pancreas-emiocytosis. ExampleComprise Biguanide derivative (for example, melbine (metformin)), sulfonylurea (SU) (for example, orinase(tolbutamide), chlorpropamide (chlorpropamide), tolazamide (tolazamide), Acetohexamide(acetohexamide), the chloro-N-[(1-Pyrrolizidine of 4-base amino) carbonyl]-benzsulfamide (Ge Longping urea(glycopyramide)), glibenclamide (glyburide), gliclazide (gliclazide), 1-butyl-3-metanilylUrea, invenol (carbutamide), Glibornuride (glibonuride), Glipizide (glipizide), gliquidone(gliquidone), Ge Liepate (glisoxepid), Glybuthiazole (glybuthiazole), glybuzole(glibuzole), lattice be listed as urea (glyhexamide), glidiazine (glymidine), glypinamide (glypinamide),Phenbutamide (phenbutamide) and glycyclamide (tolylcyclamide) or its pharmaceutically acceptable salt. Other is realExample comprises that phenylalanine derivative (for example, has nateglinide (nateglinide) [N-(trans-4-isopropyl basic ring of following formulaHexyl carbonyl)-D-phenylalanine] (referring to EP196222 and EP526171)

Repaglinide [(S)-2-ethyoxyl-4-{2-[[3-methyl isophthalic acid-[2-(1-piperidyl) phenyl] butyl] amino]-2-Oxoethyl } benzoic acid] (referring to EP589874, EP147850A2 especially the 61st page of embodiment 11, and EP207331A1); (2S)-2-benzyl-3-(cis-six hydrogen-2-iso-dihydro-indole-group carbonyl)-calcium propionate dihydrate (for example, Mitiglinide(mitiglinide) (referring to EP507534)) and Glimepiride (glimepiride) (referring to EP31058).

Other example of second activating agent that can use with peptide of the present invention and polypeptides in combination comprise DPP-IV inhibitor,GLP-1 and GLP-1 activator.

DPP-IV is responsible for making GLP-1 to deactivate. More specifically, GLP-1 produces GLP-1 receptor antagonist, thereby shortensTo the physiological responses of GLP-1. GLP-1 be pancreas insulin secretion mainly irritate thing and to glucose, processing has directly usefulEffect.

DPP-IV (DPP IV) inhibitor can be peptide, or is preferably non-peptide. DPP-IV inhibitor is every kind of feelingsUnder shape, briefly and be particularly disclosed in for example WO98/19998, DE19616486A1, WO00/34241 and WO95/In 15309, in each situation, be particularly disclosed in the end product of compound claim and work embodiment, finally produceOwner's topic, pharmaceutical preparation and claim are all incorporated in the application to quote these disclosed modes at this. Preferably distinguishSpecifically be disclosed in those compounds of the embodiment 3 of WO98/19998 and the embodiment 1 of WO00/34241.

GLP-1 (glucagon-like-peptide-1) is pancreotropic hormone albumen, and it is set forth in the people such as such as W.E.Schmidt,Diabetologia, 28,1985,704-707 and US5, in 705,483.

Term " GLP-1 activator " comprises GLP-1 (7-36) NH₂Variant and analog, they are specifically disclosed in US5,120,712, US5,118666, US5,512,549, WO91/11457 and C.Orskov etc., J.Biol.Chem.264In (1989) 12826. Other example comprises GLP-1 (7-37), wherein in this compound, and Arg³⁶Carboxyl terminal amide functionalGroup is by GLP-1 (7-36) NH₂The Gly of 37 of molecule replaces, and variant and analog, comprises GLN⁹-GLP-1(7-37)、D-GLN⁹-GLP-1 (7-37), acetyl group LYS⁹-GLP-1(7-37)、LYS¹⁸-GLP-1 (7-37), and GLP-1 (7-especially37)OH、VAL⁸-GLP-1(7-37)、GLY⁸-GLP-1(7-37)、THR⁸-GLP-1(7-37)、MET⁸-GLP-1 (7-37) and 4-Imidazo propiono-GLP-1. Especially preferred be by people such as Greig at Diabetologia1999, in 42,45-50, describeGLP activator analog exendin-4.

" antidiabetic " definition also comprises insulin sensitivity enhancer, its can recover impaired insulin receptor function withReduce insulin resistance and therefore strengthen insulin sensitivity. Example comprises hypoglycemic thiazolidinediones derivative (for example, latticeRow ketone (glitazone), (S)-((3,4-dihydro-2-(phenyl-methyl)-2H-1-chromene-6-yl) methyl-thiazolidine-2,4-diketone (Englitazone (englitazone)), 5-{[4-(3-(5-methyl-2-phenyl-4-Azoles base)-1-oxo thirdBase)-phenyl]-methyl }-thiazolidine-2,4-diketone (Darglitazone (darglitazone)), 5-{[4-(1-methyl-cyclohexyl base)Methoxyl group)-phenyl] methyl }-thiazolidine-2,4-diketone (Ciglitazone (ciglitazone)), 5-{[4-(2-(1-indyl)Ethyoxyl) phenyl] methyl }-thiazolidine-2,4-diketone (DRF2189), 5-{4-[2-(5-methyl-2-phenyl-4-Azoles base)-Ethyoxyl)] benzyl }-thiazolidine-2,4-diketone (BM-13.1246), 5-(2-naphthyl sulfonyl)-thiazolidine-2,4-diketone(AY-31637), two { 4-[(2,4-dioxo-5-thiazolidinyl) methyl] phenyl } methane (YM268), 5-{4-[2-(5-firstBase-2-phenyl-4-Azoles base)-2-hydroxyl-oxethyl] benzyl }-thiazolidine-2,4-diketone (AD-5075), 5-[4-(1-benzeneBase-1-cyclopropane carbonyl amino)-benzyl]-thiazolidine-2,4-diketone (DN-108) 5-{[4-(2-(2,3-indoline-1-Base) ethyoxyl) phenyl] methyl }-thiazolidine-2,4-diketone, 5-[3-(the chloro-phenyl of 4-])-2-propynyl]-5-phenyl sulphonylBase) thiazolidine-2,4-diketone, 5-[3-(4-chlorphenyl])-2-propynyl]-5-(4-fluorophenyl-sulfonyl) thiazolidine-2,4-Diketone, 5-{[4-(2-(methyl-2-pyridine radicals-amino)-ethyoxyl) phenyl] methyl }-thiazolidine-2,4-diketone (Rosiglitazone(rosiglitazone)), 5-{[4-(2-(5-ethyl-2-pyridine radicals) ethyoxyl) phenyl]-methyl thiazolidine-2,4-diketone(Pioglitazone (pioglitazone)), 5-{[4-((3,4-dihydro-6-hydroxyl-2,5,7,8-tetramethyl-2H-1-benzo pyrroleMutter-2-yl) methoxyl group)-phenyl]-methyl }-thiazolidine-2,4-diketone (troglitazone (troglitazone)), 5-[6-(2-Fluoro-benzyl oxygen base) naphthalene-2-ylmethyl]-thiazolidine-2,4-diketone (MCC555), 5-{[2-(2-naphthyl)-benzoAzoles-5-Base]-methyl } thiazolidine-2,4-diketone (T-174) and 5-(2,4-dioxo thiazolidine-5-ylmethyl)-2-methoxyl group-N-(4-Trifluoromethyl-benzyl) benzamide (KRP297)).

Other antidiabetic comprises insulin signaling pathway adjusting control agent, as Protein-tyrosine-phosphatase (PTPases) presses downPreparation, the non-small molecule mimetics compound of anti-diabetic and glutamine-fructose-6-phosphate acylamino-transferase (GFAT) suppressAgent; The compound of the Hepatic glucose production of impact imbalance, as G-6-Pase (G6Pase) inhibitor, fructose-1,6-is twoPhosphatase (F-1,6-Bpase) inhibitor, glycogen phosphorylase (GP) inhibitor, glucagon receptor antagonist and phosphoric acid alkeneAlcohol pyruvic acid carboxylic kinases (PEPCK) inhibitor; Pyruvic dehydrogenase kinase (PDHK) inhibitor; The inhibitor of gastric emptying; Pancreas isletElement; GSK-3 inhibitor; Retinoid X acceptor (RXR) activator; β-3AR activator; UCPS (UCPs) activator; Non-Lattice row ketone type PPAR gamma agonist; Dual PPAR α/PPAR gamma agonist; Anti-diabetic vanadium-containing compound; Duodenin(incretin) hormone, as hyperglycemic factor-sample peptide-1 (GLP-1) and GLP-1 activator; Beta cell imidazoline receptor antagonismAgent; Miglitol (miglitol); α₂-adrenaline antagonist; And pharmaceutically acceptable salt.

In one embodiment, the invention provides combination product, particularly pharmaceutical combination product, it comprises treatment effectivelyAmount according to the defined polypeptide of arbitrary formula or its acid amides, ester or salt or its bioconjugates in formula I to IV, and one or moreBe selected from following therapeutic activity agent: B-adrenergic receptor blocking agent, for example acebutolol, atenolol, betaxolol, ratioSuo Luoer, metoprolol, Nadolol, Propranolol, Sotalol and timolol; Angiotensin II receptor antagonists,For example AT1 blocking agent; Antidiabetic, for example DPPIV inhibitor (for example vildagliptin (vildagliptin)) and GLP-1 peptideActivator.

Term " anoretic " comprises lipase inhibitor (for example, orlistat (orlistat)) and appetite inhibitor (exampleAs, sibutramine (sibutr amine) and Phentermine (phentermine)).

Aldosterone synthase inhibitors or its pharmaceutically acceptable salt should be understood to have inhibition aldosterone and produceThe active component of character. Aldosterone synthase (CYP11B2) is the final step that catalysis aldosterone produces in adrenal cortexThe mitochondrial cytochrome P450 enzyme of (, 11-deoxycorticosterone is to the conversion of aldosterone). Knownly use so-called aldosterone synthaseThe inhibition that inhibitor produces aldosterone is treatment hypopotassaemia, hypertension, congestive heart failure, auricular fibrillation or kidney failureSuccessful alternative. This aldosterone synthase suppresses activity can be by those skilled in the art according to standard analysis (for example,US2007/0049616) easily measure.

Such aldosterone synthase inhibitors comprises steroidal and non-steroidal aldosterone synthase inhibitors, and the latter is preferred.

The preferably aldosterone synthase inhibitors of commercially available aldosterone synthase inhibitors or those health authority's approvals.

Such aldosterone synthase inhibitors comprises the compound with different structure feature. Non-steroidal aldosterone synthase suppressesThe example of agent is (+)-enantiomter (US patent 4617307 Hes of Arensm (fadrozole) hydrochloride of following formula4889861)

Or, if suitable, its pharmaceutically acceptable salt.

The aldosterone synthase inhibitors that can be used for described combination product is conventionally and to be specifically disclosed in for example US2007/Compound in 0049616 and analog, be especially disclosed in the end product of compound claim and work embodiment,Theme, pharmaceutical preparation and the claim of end product this by reference this disclosed mode be incorporated in the application. Be suitable forInclude but not limited to 4-(6,7-dihydro-5H-pyrrolo-[1,2-c] imidazoles-5-in preferred aldosterone synthase inhibitors of the present inventionBase)-3-methyl benzonitrile; 5-(the chloro-4-cyano-phenyl of 2-)-6,7-dihydro-5H-pyrrolo-[1,2-c] imidazoles-5-formic acid (4-firstOxy-benzyl) methyl nitrosourea; 4 '-fluoro-6-(6,7,8,9-tetrahydrochysene-5H-imidazo [1,5-a] azepine-5-yl) biphenyl-3-firstNitrile; 5-(4-cyano group-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo-[1,2-c] imidazoles-5-formic acid butyl ester; 4-(6,7-Dihydro-5H-pyrrolo-[1,2-c] imidazoles-5-yl)-2-HOMOVERATRONITRILE; 5-(the chloro-4-cyano-phenyl of 2-)-6,7-dihydro-5H-Pyrrolo-[1,2-c] imidazoles-5-formic acid 4-luorobenzyl ester; 5-(4-cyano group-2-Trifluoromethoxyphen-l)-6,7-dihydro-5H-pyrroleCough up also [1,2-c] imidazoles-5-formic acid methyl ester; 5-(4-cyano group-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo-[1,2-C] imidazoles-5-formic acid 2-isopropoxy ethyl ester; 4-(6,7-dihydro-5H-pyrrolo-[1,2-c] imidazoles-5-yl)-2-methyl benzylNitrile; 4-(6,7-dihydro-5H-pyrrolo-[1,2-c] imidazoles-5-yl)-3-fluorine benzonitrile; 4-(6,7-dihydro-5H-pyrrolo-[1,2-C] imidazoles-5-yl)-2-HOMOVERATRONITRILE; The fluoro-4-of 3-(7-methylene-6,7-dihydro-5H-pyrrolo-[1,2-c] imidazoles-5-Base) benzonitrile; The fluoro-4-[7-of cis-3-(the fluoro-benzyl of 4-)-5,6,7,8-tetrahydrochysene-imidazo [1,5-a] pyridine-5-yl] benzonitrile;4 '-fluoro-6-(9-methyl-6,7,8,9-tetrahydrochysene-5H-imidazo [1,5-a] azepine-5-yl) biphenyl-3-formonitrile HCN; 4 '-fluoro-6-(9-methyl-6,7,8,9-tetrahydrochysene-5H-imidazo [1,5-a] azepine-5-yl) biphenyl-3-formonitrile HCN, or under each situation its(R) or (S) enantiomter; Or if desired, its pharmaceutically acceptable salt.

Term aldosterone synthase inhibitors also comprises and is disclosed in WO2008/076860, WO2008/076336, WO2008/076862, compound and the class in WO2008/027284, WO2004/046145, WO2004/014914, WO2001/076574Like thing.

In addition, aldosterone synthase inhibitors also comprise following in disclosed compound and analog: U.S. patent applicationUS2007/0225232、US2007/0208035、US2008/0318978、US2008/0076794、US2009/0012068、US20090048241 and PCT application WO2006/005726, WO2006/128853, WO2006128851, WO2006/128852,WO2007065942, WO2007/116099, WO2007/116908, WO2008/119744 and European patent application EP1886695. Be applicable to preferred aldosterone synthase inhibitors of the present invention and include but not limited to the 8-(4-being developed by SpeedelFluorophenyl)-5,6-dihydro-8H-imidazo [5,1-c1[1,41Piperazine; 4-(5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-yl)-2-fluorine benzonitrile; 4-(5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-yl)-2,6-difluoro benzylNitrile; 4-(5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-yl)-2-HOMOVERATRONITRILE; 3-(5,6-dihydro-8H-Imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-yl) benzonitrile; 4-(5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-Base) phthalonitrile; 4-(8-(4-cyano-phenyl)-5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-yl) benzonitrile; 4-(5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-yl) benzonitrile; 4-(5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine-8-yl) naphthalene-1-formonitrile HCN; 8-[4-(1H-TETRAZOLE-5-yl) phenyl 1-5,6-dihydro-8H-imidazo [5,1-c] [Isosorbide-5-Nitrae]Piperazine; Or under each situation its (R) or (S) enantiomter; Or if desired, its pharmaceutically acceptable salt.

The aldosterone synthase inhibitors that can be used for this combination product is generally and to be particularly disclosed in for example WO2009/156462 and WO2010/130796 in compound and analog, be especially disclosed in compound claim and embodimentEnd product, in theme, pharmaceutical preparation and the claim of end product. The preferred aldehydes sterone that is applicable to combination of the present invention closesEnzyme inhibitor comprises 3-(the fluoro-3-methyl-2-of 6-pyridin-3-yl-1H-indoles-1-ylmethyl)-benzonitrile hydrochloride, 1-(4-methaneSulfonyl-benzyl)-3-methyl-2-pyridin-3-yl-1H-indoles, the chloro-1-methyl of 2-(5-benzyloxy-pyridin-3-yl)-6--1H-indoles, 5-(3-cyano group-1-Methyl-1H-indole-2-yl)-nicotinic acid ethyl ester, N-[5-(the chloro-3-cyano group-1-of 6-methyl-1H-indoles-2-yl)-pyridin-3-yl methyl]-ethyl sulfonamide, pyrrolidines-1-sulfonic acid 5-(the chloro-3-cyano group-1-of 6-methyl isophthalic acid H-Indoles-2-yl)-pyridin-3-yl ester, N-methyl-N-[5-(1-Methyl-1H-indole-2-yl)-pyridin-3-yl methyl]-first sulphurAcid amides, the chloro-1-methyl-2-{5-[(2-of 6-pyrrolidin-1-yl-ethylamino)-methyl]-pyridin-3-yl }-1H-indoles-3-firstNitrile, the chloro-2-[5-of 6-(4-methane sulfonyl-piperazine-1-ylmethyl)-pyridin-3-yl]-1-Methyl-1H-indole-3-formonitrile HCN, 6-Chloro-1-methyl-2-{5-[(1-methyl-piperidin-4-yl amino)-methyl]-pyridin-3-yl }-1H-indole-3-formonitrile, morpholine-4-formic acid [5-(the chloro-3-cyano group-1-of 6-Methyl-1H-indole-2-yl)-pyridin-3-yl methyl]-acid amides, N-[5-(the chloro-1-of 6-Methyl-1H-indole-2-yl)-pyridin-3-yl methyl]-ethyl sulfonamide, C, C, the fluoro-N-[5-of C-tri-(1-Methyl-1H-indole-2-Base)-pyridin-3-yl methyl]-Methanesulfomide, N-[5-(the chloro-4-cyano group-phenyl of 3-)-pyridin-3-yl]-4-trifluoromethyl-benzeneSulfonamide, N-[5-(the chloro-4-cyano group-phenyl of 3-)-pyridin-3-yl]-1-phenyl-Methanesulfomide, N-(5-(the chloro-4-cyano group of 3-benzeneBase) pyridin-3-yl) butane-1-sulfonamide, N-(1-(5-(4-cyano group-3-methoxyphenyl) pyridin-3-yl) ethyl) second sulphonylAmine, N-((5-(the chloro-4-cyano-phenyl of 3-) pyridin-3-yl) (cyclopropyl) methyl) ethyl sulfonamide, N-(cyclopropyl (5-(1H-YinDiindyl-5-yl) pyridin-3-yl) methyl) ethyl sulfonamide, N-(cyclopropyl (5-naphthalene-1-base-pyridin-3-yl) methyl) ethyl sulfonamide,Ethyl sulfonic acid [5-(the chloro-1-methyl isophthalic acid of 6-H-pyrrolo-[2,3-b] pyridine-2-yl)-pyridin-3-yl methyl]-acid amides and ethyl sulfonic acid{ [5-(the chloro-4-cyano group-phenyl of 3-)-pyridin-3-yl]-cyclopropyl-methyl }-ethyl-acid amides.

Term " blockade of endothelin receptors agent " comprises Bosentan and ambrisentan (ambrisentan).

Term " CETP inhibitor " refer to suppress CETP (CETP) mediation by various cholesteryl esters andTriglyceride is transported to the compound of LDL and VLDL from HDL. This type of CETP suppresses activity and is easy to by those skilled in the artFor example, measure according to standard analytical process (, U.S.Pat.No.6,140,343). The example comprises and is disclosed in U.S.Pat.No.6,Compound in 140,343 and U.S.Pat.No.6,197,786 (for example [2R, 4S] 4-[(3,5-is two-trifluoromethyl-benzyl)-Methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-Ethyl formate (torcetrapib(torcetrapib)); Be disclosed in U.S.Pat.No.6, compound (for example, (2R)-3-{[3-(the chloro-3-of 4-in 723,752Ethyl-phenoxy group)-phenyl]-[[3-(1,1,2,2-tetrafluoro-ethyoxyl)-phenyl]-methyl]-amino }-1,1, the fluoro-2-of 1-tri-Propyl alcohol); Be disclosed in U.S. Patent application the 10/807th, the compound in No. 838; Be disclosed in U.S.Pat.No.5, in 512,548Polypeptide derivative; Be disclosed in respectively J.Antibiot., 49 (8): 815-816 (1996), andBioorg.Med.Chem.Lett.; The rosenonlactone of the cholesteryl ester in 6:1951-1954 (1996)(rosenonolactone) derivative and phosphate ester-containing analog. In addition, CETP inhibitor also comprises and is disclosed in WO2000/017165、WO2005/095409、WO2005/097806、WO2007/128568、WO2008/009435、WO2009/059943 and WO2009/071509 in those.

Term " nep inhibitor " refers to the compound that suppresses neutral endopeptidase (NEP) EC3.4.24.11. Example comprises bankSha Qu (Candoxatril), Candoxatrilat (Candoxatrilat), Dexecadotril (Dexecadotril), Ecadotril(Ecadotril), racecadotril (Racecadotril), sampatrilat (Sampatrilat), fasidotril(Fasidotril), omapatrilat (Omapatrilat), gemopatrilat (Gemopatrilat), daglutril(Daglutril), SCH-42495, SCH-32615, UK-447841, AVE-0848, PL-37 and (2R, 4S)-5-biphenyl-4-Base-4-(3-carboxyl-propiono amino)-2-methyl-valeric acid ethyl ester or its pharmaceutically acceptable salt. Nep inhibitor also wrapsDraw together as US Patent No. 5,155 dipeptidase derivant of disclosed phosphono/biaryl substituted in 100. Nep inhibitor also wrapsDraw together as disclosed N-sulfydryl acylphenylalanines derivative in PCT application WO2003/104200. Nep inhibitor also comprise asThe anti-high blood of disclosed double action in PCT application WO2008/133896, WO2009/035543 or WO2009/134741Pressing. Other example comprises disclosed compound in U.S. Patent application 12/788,794,12/788,766 and 12/947,029.Nep inhibitor also comprises WO2010/136474, WO2010/136493, WO2011/061271 and U.S. Provisional Application numberDisclosed compound in 61/414171 and 61/414163.

In one embodiment, the invention provides the method for the individual apj receptor of activation, wherein the method comprises to thisPuting together according to the defined polypeptide of arbitrary formula or its acid amides, ester, salt or its biology in formula I to IV of individual administering therapeutic effective doseThing.

In one embodiment, the invention provides illness or the disease of the individual response of the activation to apj receptor for the treatment ofMethod, wherein the method comprise to this individuality administering therapeutic effective dose according to the defined polypeptide of arbitrary formula in formula I to IVOr its acid amides, ester, salt or its bioconjugates.

In one embodiment, the invention provides the illness of the individual activation to apj receptor (excitement) response for the treatment ofOr the method for disease, wherein this illness or disease are selected from acute decompensation DHF (ADHF), chronic heart failure, lungProperty hypertension, auricular fibrillation, Bu Lugeda Cotard, Ventricular Tachycardia, atherosclerotic, hypertension, ISR, officePortion's ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, cardiac arrhythmia, water retention, diabetes (comprising gestational diabetes mellitus),Obesity, peripheral arterial disease, cerebrovas-cularaccident, temporary ischemic episode, traumatic brain injury, ALS, burn(comprising sunburn) and pre-eclampsia.

In one embodiment, the invention provides according to the defined polypeptide of arbitrary formula or its biology in formula I to IV and sewCompound, it is as medicine.

In one embodiment, the invention provides suc as formula the defined polypeptide of arbitrary formula or its acid amides in I to IV, ester,Salt or its bioconjugates are in the purposes of preparing in medicine, and described medicine is used for the treatment of the illness of the activation response to apj receptorOr disease. In another embodiment, the invention provides according to the defined polypeptide of arbitrary formula or its acid amides in formula I to IV, ester,Salt or its bioconjugates purposes in the illness of activation response or the medicine of disease to apj receptor for the preparation for the treatment of,Wherein said illness or disease are selected from acute decompensation DHF (ADHF), chronic heart failure, the high blood of lung especiallyPressure, auricular fibrillation, Bu Lugeda Cotard, Ventricular Tachycardia, atherosclerotic, hypertension, ISR, ischaemicProperty angiocardiopathy, cardiomyopathy, cardiac fibrosis, cardiac arrhythmia, water retention, diabetes (comprising gestational diabetes mellitus), obesity,Peripheral arterial disease, cerebrovas-cularaccident, temporary ischemic episode, traumatic brain injury, ALS, burning (comprises solarizationWound) and pre-eclampsia.

Example of the present invention: peptide and polypeptide are synthetic

Described peptide synthesizes by standard solid-phase Fmoc chemistry. Described peptide is at Prelude^TMPeptide synthesizer (ProteinTechnologies, Inc., Tucson, USA) upper assembling. The peptide on C-end with free carboxy acid is from 2-chlorine triphen firstThe chloro-PS-resin of base (ABCR, Karlsruhe, Germany) is synthetic. The peptide on C-end with the mono-substituted formamide of N-isSynthetic from being loaded with the BAL-AM-PS-resin (EMCMicrocollections, T ü bingen, Germany) of amine.

Described peptide carrys out purifying by preparative reversed-phase HPLC. Use with lower prop:

● WatersSunFirePrepC18OBD post, 5 μ m, 30x100mm, part number 186002572 (post orTwo columnss in series)

● WatersAtlantisPrepOBDT3 post, 5 μ m, 30x150mm, part number .186003703

Mobile phase by eluant, eluent A (at H₂0.1%TFA in O) and eluant, eluent B (ACN) composition. Based on the tool of separation problemBody requires to design gradient. By pure product from ACN/H₂Freeze-drying in O.

Detect at λ=214nm and UPLC-MS electron spray ionisation and carry out dividing of product with UV by HPLCAnalyse.

In table 4, illustrated peptide is to synthesize with general procedure hereinafter described. Unsubstituted N-or C-endRespectively by italic H-or-OH represents.

Table 4:

Embodiment	Sequence
		Embodiment 1	pE-R-P-R-C-S-H-K-G-P-Nle-C-F-OH 49 -->
Embodiment 2	pE-R-P-R-hC-S-H-K-G-P-(D-Nle)-C-y-OH
		Embodiment 3	pE-R-P-R-L-S-C-K-G-P-Nle-C-F-OH
Embodiment 4	pE-R-P-R-L-S-C-K-G-P-Nle-(D-hC)-F-OH
		Embodiment 5	pE-R-P-R-L-S-c-K-G-P-Nle-(D-hC)-F-OH
Embodiment 6	pE-R-P-R-L-S-hC-K-G-P-Nle-(D-hC)-F-OH
		Embodiment 7	pE-R-P-R-L-S-(D-hC)-K-G-P-Nle-(D-hC)-F-OH
Embodiment 8	pE-R-P-R-L-S-(D-hC)-K-G-P-Nle-C-F-OH
		Embodiment 9	pE-R-P-R-L-S-(D-hC)-K-G-P-Nle-hC-F-OH
Embodiment 10	pE-R-P-R-L-S-(D-hC)-K-G-P-Nle-C-OH
		Embodiment 11	pE-R-P-R-L-S-C-K-G-P-Nle-c-F-OH
Embodiment 12	pE-R-P-R-L-S-C-K-G-P-Nle-hC-F-OH
		Embodiment 13	pE-R-P-R-L-S-hC-K-G-P-Nle-hC-F-OH
Embodiment 14	pE-R-P-R-L-S-H-K-C-P-Nle-C-F-OH
		Embodiment 15	PE-R-P-R-L-S-Aib-K-C-P-Nle-C-NH (phenethyl)
Embodiment 16	pE-R-P-R-L-S-a-K-C-P-Nle-C-f-OH
		Embodiment 17	pE-R-P-R-L-a-H-K-C-P-Nle-C-f-OH
Embodiment 18	Piperidines-4-formyl-R-P-R-L-a-H-K-C-P-Nle-C-f-OH
		Embodiment 19	pE-R-P-R-L-a-Aib-K-C-P-Nle-C-f-OH
Embodiment 20	pE-R-P-R-L-S-Aib-K-C-P-Nle-C-f-OH
		Embodiment 21	pE-R-P-R-L-s-H-K-C-P-Nle-C-f-OH

Wherein use two amino acid represents of " * " mark to form the amino acid of disulphide by their side chain.

Analytical method

● 1a) HPLC-analytical method A

● post: WatersXbridge^TMC18 (50x4.0mm), 3.5 μ m; Part number: 186003031

● eluant, eluent A: the 0.07%TFA/ eluant, eluent B in water: the 0.1%TFA in ACN

● flow velocity: 1.5ml/min

● temperature: 40 DEG C

● gradient:

Time [min]	A[％]	B[％]
			0.0	95	5
10.0	0	100
			12.0	0	100
12.2	95	5

1b) HPLC-analytical method B

● post: XBridgeBEH300C18 (100x4.6mm), 3 μ m; Part number: 186003612

● eluant, eluent A: the 0.1%TFA/ eluant, eluent B in water: the 0.1%TFA in ACN

● flow velocity: 1.0ml/min

● temperature: 40 DEG C

● gradient:

Time [min]	A[％]	B[％]
			0.0	98	2
18	2	98
			20	2	98
22	98	2

2a) UPLC-MS-analytical method C

● post: AcquityBEH300C18 (50x2.1mm), 1.7 μ m; Part number: 186003685

● eluant, eluent A: the 0.05%TFA/ eluant, eluent B in water: the 0.04%TFA in ACN

● flow velocity: 1.0ml/min

● temperature: 80 DEG C

● gradient:

Time [min]	A[％]	B[％]
			0.0	100	0
0.2	100	0
			4.4	2	98
4.8	2	98
			4.9	100	0
5.0	100	0

2b) UPLC-HRMS-analytical method D

●WatersAcquityBEHC18,1.7 μ m, 2.1x50mm; Part number: 186002350

● eluant, eluent A: the 0.05%FA+3.75mM ammonium acetate in water; Eluant, eluent B: the 0.04%FA in ACN

● flow velocity: 1.0ml/min

● temperature: 50 DEG C

● gradient: 2 to 98%, go through 4.4min

The analysis data of the peptide of embodiment 1 to 21 are summarised in table 5, and adopt the analytical method generation in upper description.

Table 5:

General synthesis program

1) first amino acid of load to 2-chlorine trityl chloride resin and Fmoc-remove

Fully wash 2-chlorine trityl chloride resin (1eq., 1.0-1.6mmol/g) with DCM. The amino acid of expecting is (logicalOften, with respect to the 0.5-2eq. of resin, be considered as 1.6mmol/g load) be dissolved in DCM (about 10mL/ gram resin) and DIPEA (relativelyBe 4eq. in resin, be considered as 1.6mmol/g load) in. This solution is added in resin and at room temperature by suspension jolting3-16h. Outwell the liquid in resin, then use successively DCM/MeOH/DIPEA (17:2:1), DCM, DMA, DCM fully to wash.

For the mensuration of removal and the load of Fmoc, by resin and piperidines/DMA (1:4) or 4-methyl piperidine/DMA (1:4) (12x10mL/ gram of initial resin) jolting use DMA (2x10mL/ gram of initial resin) washing repeatedly. Dilute and close with MeOHAnd solution to V/ gram of initial resin of 250mL volume. With MeOH by the 2mL aliquot (V of this solution_a) be further diluted to250ml(V_t). For MeOH reference, measure the UV trap at 299.8nm place, degree of being absorbed A. Use successively DMA, DCM, DMA,The abundant washing resin of DCM is also dry in high vacuum at 40 DEG C, obtains mg resin.

Calculate the load of resin according to following formula:

Load [mol/g]=(AxV_txV)/(dxεxV_axm)

(wherein, d: cuvette width; ε=7800Lmol^-1cm^-1)

2) at Prelude^TMSolid-phase peptide on synthesizer is synthetic

2a) synthetic circulation A

Use DMA washing resin. Remove Fmoc with piperidines/DMA (1:4) or 4-methyl piperidine/DMA (1:4) reprocessing.Use DMA washing resin. Carry out in the following manner coupling: add Fmoc-amino acid (3eq.; 0.3M solution in NMP),HCTU (3eq.; 0.3M solution in NMP) and DIPEA (4.5eq.; 0.9M solution in NMP), at room temperature exist subsequentlyUnder nitrogen, this suspension is mixed, conventionally mix 15 minutes to 4 hours, this depends on real needs. With after DMA washing, logicalNormal repetition coupling step 1 to 3 time, this depends on real needs. After washing with DMA, by adding Ac₂O/ pyridine/DMA (1:Mixture 1:8) also at room temperature mixes subsequently this suspension and implements end-blocking. Resin is washed with DMA.

2b) synthetic circulation B

Use DMA washing resin. Remove Fmoc with piperidines/DMA (1:4) or 4-methyl piperidine/DMA (1:4) reprocessing.Use DMA washing resin. Carry out in the following manner coupling: add Fmoc-amino acid (3eq.; 0.2M solution in NMP),HCTU (3eq.; 0.3M solution in NMP) and DIPEA (4.5eq.; 0.9M solution in NMP), at room temperature exist subsequentlyUnder nitrogen, this suspension is mixed, conventionally mix 15 minutes to 4 hours, this depends on real needs. With after DMA washing, logicalNormal repetition coupling step 1 to 3 time, this depends on real needs. After washing with DMA, by adding Ac₂O/ pyridine/DMA (1:Mixture 1:8) also at room temperature mixes subsequently this suspension and implements end-blocking. Resin is washed with DMA.

3) from resin cracking and follow blocking group remove

3a) cleavage method A

At room temperature by resin (0.1mmol), (2-3mL) jolting 2 is little with 95%aq.TFA/EDT/TIS (95:2.5:2.5)Time. Leach cracked solution, and add fresh solution (2-3mL). At room temperature jolting suspension 1 hour, then leaches crackingSolution. Add fresh solution (2-3mL) jolting suspension 1 hour at room temperature. Leach cracked solution. By the cracking mergingThe mixture (1:1) that solution is poured onto cold heptane/ether lentamente (35mL) in, be precipitated. Centrifugal suspension also pours outSupernatant. With (10-20mL) wash residual thing of cold heptane/ether (1:1), centrifugal suspension also pours out supernatant. By this stepSuddenly carry out 1-2 time. Drying solid in high vacuum.

4) cyclization method

4a) cyclization method A (disulphide formation)

The linear precursor peptide of complete deprotection is dissolved in to H₂O/DMSO (9:1) or (4:1) in, obtain common 0.5-The concentration of 7mM. Then at room temperature reactant mixture is stirred to 16-96 hour conventionally, this depends on demand, and then at heightIn vacuum, be concentrated into dry.

To the synthetic of representative embodiment be described hereinafter.

Embodiment 1pE-R-P-R-C*-S-H-K-G-P-Nle-C*-F-OH (disulphide C⁵-C¹²) synthetic

Embodiment 1

● the preparation of intermediate 1a

(being loaded to the mensuration of the load capacity of 2-chlorine trityl chloride resin, Fmoc removal and resin with Fmoc-F-OH)

By DCM (3x) washing for 2-chlorine trityl chloride resin (10.0g, 16.0mmol). Add Fmoc-F-OHThe DCM (100mL) of (12.4g, 32.0mmol) and the solution of DIPEA (11.2mL, 64.0mmol) by suspension at room temperatureJolting 5h. By DCM/MeOH/DIPEA for resin (17:2:1) (3x), DCM (3x), DMA (3x), DCM (3x) wash fully.

Then resin is processed to 12 times (2min) with the mixture (1:4) (50mL is each) of piperidines/DMA and then use DMA(2x) washing. Collect piperidines/DMA solution and DMA wash solution, for measuring load capacity (referring to general procedure). Resin is usedDCM (3x), DMA (3x), DCM (3x) wash and the dry intermediate 1a (12.8g that obtains under vacuum fully; Load capacity=0.79mmol/g)。

● the preparation of intermediate 1b

(linear peptides assembling)

At Prelude^TMOn peptide synthesizer, making intermediate 1a (127mg, 0.10mmol) carry out solid-phase peptide synthesizes. Carry out as followsCoupling:

Coupling	AA	The x reaction time is counted in coupling	Synthetic circulation
				1	C(Trt)	2x 45min	A
2	Nle	2x 45min	A
				3	P	2x 45min	A
4	G	2x 90min	A
				5	K(Boc)	2x 45min	A
6	H(Trt)	2x 45min	A
				7	S(tBu)	1x 3h	A
8	C(Trt)	2x 45min	A
				9	R(Pbf)	4x 1h	A
10	P	2x 90min	A
				11	R(Pbf)	4x 1h	A
12	pE	2x 90min	A

● the preparation of intermediate 1c

(from resin cracking and follow blocking group remove)

Intermediate 1b (0.10mmol) is washed carefully with DCM (4x). Add 95%aq.TFA/EDT/TIS (95:Mixture (2mL) 2.5:2.5), and by room temperature jolting 2h of suspension. Leach cracked solution, and add fresh crackingSolution (2mL). By suspension at room temperature jolting 1h then leach cracked solution. Add fresh solution (2mL) and by suspendibleLiquid is jolting 1h at room temperature. Leach cracked solution. By the 95%TFA aqueous solution (1mL) washing for resin, also leached and receivedCollection. The mixture (1:1) that the cracked solution of merging is poured onto to cold heptane/ether (10mL) in, be precipitated. By mixture fromThe heart also pours out supernatant. By (10mL) washing again of cold heptane/ether (1:1) for solid, by centrifugal mixture and topple overGo out supernatant. By solid dry intermediate 1c that obtains under vacuum.

● the preparation of embodiment 1

(cyclisation and purifying)

Intermediate 1c is dissolved in to H₂O/DMSO (4:1) (4mL) in. By reactant mixture at stirring at room temperature 64h. To mixThing is committed to preparation HPLC, and by pure fraction from ACN/H₂Freeze-drying in O, obtains embodiment 1, is white solid (38.5mg;0.019mmol)。

By analytic type HPLC (analytical method A; t_R=3.13min) and UPLC-MS (analytical method C; Measured value: [M+3]³⁺=503.9; Calculated value: [M+3]³⁺=503.9) analyze this pure products.

Embodiment 2pE-R-P-R-hC*-S-H-K-G-P-(D-Nle)-C*-y-OH's is synthetic

Peptide on solid carrier is synthetic:

By standard Fmoc Fmoc-D-Tyr (tBu) chlorine trityl resin (replacement: 1.1mmol/g) carry out hand for chemistryWork solid-phase peptide is synthetic. By 0.3mmol resin in DMF swelling 30 minutes; Venting DMF, and resin is molten with the DMF of 20% piperidinesLiquid is processed 30min to remove Fmoc group. By DMF washing for resin 3 times, and with preactivated Fmoc Freamine Ⅲ (FmocAmino acid/HBTU/HOBt/NMM=3:3:3:6eq) coupling 2 hours. After each coupling, carry out ninhydrin test, to check couplingEfficiency.

Amino acid by repeating to remove Fmoc protecting group and coupling protection is until N-end is assembled peptide chain on resin.

After last amino acid coupling, by DMF and ether washing for peptide resin, and vacuum drying. By dry peptide treeTFA cleavage mixture (TFA/ thioanisole/phenol/EDT/H for fat₂O=87.5:5:2.5:2.5:2.5, v/v) process, forCracking is also removed Side chain protective group. Thick peptide is precipitated from cold ether, collect and be dried under high vacuum by filtration. Thick peptide is existedHPLC (post: 2 "-inch DeltaPakC18, wavelength: 215nm) upper purifying, obtain the product needing.

Cyclisation:

Each thick peptide is dissolved in to water-acetonitrile (A.C.S. reagent, Fisher) (approximately 80%:20% with the concentration of 1mg/mL;Water: acetonitrile, V:V) in, under vigorous stirring by 0.1MI₂The 50%AcOH of (A.C.S. reagent, SigmaAldrich)(A.C.S. reagent, Fisher)/H₂O solution is added drop-wise in described solution, until I₂Color continues. Once be oxidized (by dividingAnalyse type HPLC and mass spectrography monitoring), continuing to drip 1ML-ascorbic acid (A.C.S. reagent, SigmaAldrich) under stirringThe aqueous solution, to reduce excessive I₂, until solution becomes colorless. After filtration, solution is above uploaded to 2-inch C18 postUpper (detecting at 215nm), adopts TFA buffer solution (buffer A, 0.1%TFA (A.C.S. level, NuGenerationTechnology, LLC) the aqueous solution; Buffer B, 100% acetonitrile) purifying, by what collect > the fraction freeze-drying of 95% purity.

Embodiment 15pE-R-P-R-L-S-Aib-K-C*-P-Nle-C*-NH (phenethyl) (disulphide C⁹-C¹²) closeBecome

Embodiment 15

● the preparation of intermediate 15a

(linear peptides assembling)

At Prelude^TMOn peptide synthesizer, making phenyl ethylamine-BAL-PS-resin (167mg, 0.100mmol) carry out solid-phase peptide closesBecome. Carry out as follows coupling:

● the preparation of intermediate 15b

(from resin cracking and follow blocking group remove, then purifying)

Intermediate 15a (0.10mmol) is washed carefully with DCM (4x). Add 95%aq.TFA/EDT/TIS (95:Mixture (2mL) 2.5:2.5), and by room temperature jolting 2h of suspension. Leach cracked solution, and add fresh crackingSolution (2mL). By suspension at room temperature jolting 1h then leach cracked solution. Add fresh solution (2mL) and by suspendibleLiquid is jolting 2h at room temperature. Leach cracked solution. By the 95%TFA aqueous solution (1mL) washing for resin, also leached and receivedCollection. The mixture (1:1) that the cracked solution of merging is poured onto to cold heptane/ether (35mL), is precipitated. Mixture is centrifugalAnd pour out supernatant. By (5mL) washing again of cold heptane/ether (1:1) for solid, by centrifugal mixture and pour outClear liquid. Solid is dry under vacuum. By preparation HPLC purification of crude product freeze-drying, acquisition intermediate 15b (26.6mg,0.014mmol)。

● the preparation of embodiment 15

(cyclisation and purifying)

Intermediate 15b (26.6mg, 0.014mmol) is dissolved in to H₂O/DMSO (9:1) (13mL) in. By reactant mixtureStir at room temperature 48h. Further add DMSO (1.6mL) and at room temperature continue to stir 16h. Mixture is committed to preparationType HPLC and by pure fraction from ACN/H₂Freeze-drying in O, obtains embodiment 15, is white solid (12.8mg; 0.007mmol).

By analytic type HPLC (analytical method A; t_R=4.12min) and UPLC-MS (analytical method C; Measured value: [M+3]³⁺=490.6; Calculated value: [M+3]³⁺=490.6) analyze this pure products.

Other embodiment is synthetic in a similar manner:

● embodiment 3 to 14 and 16 to 21 is synthetic in mode similar to Example 1.

Have been found that polypeptide under embodiment has about 0.01nM to about 1100nM scope for apj receptor effectEC₅₀Value. Have been found that polypeptide under embodiment have higher than 2 minutes, higher than 5 minutes, higher than 10 minutes, higher than 20 minutes,Higher than 50 minutes with higher than the plasma stability of 60 minutes.

Can see, polypeptide of the present invention can be used as the activator of apj receptor, and therefore can be used for treatment to apj receptorDisease and the patient's condition of activation response, for example disclosed disease in literary composition.

In addition, the half-life of these peptides can comprise according to the peptide of any same form in formula I to IV or polypeptide by formation andThe bioconjugates of for example human serum albumins of the part of prolong half-life or Fc domain and further being extended.

So describe exemplary of the present invention, should have been noted by those skilled in the art, these public affairsIt is only exemplary opening content, can carry out within the scope of the invention various other and replace, adapts to and amendment. Therefore, the present inventionThe specific embodiments that is not limited to enumerate in literary composition.

Claims

1. there is the ring type polypeptide of formula I below:

X1-R-P-R-X5-X6-X7-K-X9-P-X11-X12-X13

I

Wherein:

X5 is that L or X5 are selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form disulfide bond;

X6 is S, s or a;

X7 is H, Aib or a; Or X7 is selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form twoSulfide linkage;

X9 is that G or X9 are selected from C, c, hC and D-hC; Wherein the side chain of C, c, hC or D-hC and the side chain of X12 form disulfide bond;

Wherein in X5, X7 or X9 only one be selected from C, c, hC and D-hC;

X11 is D-Nle, Nle, M or f; And

X12 is selected from C, c, hC, D-hC; The wherein C of one of the side chain of C, c, hC or D-hC and X5, X7 or X9, c, hC or D-hCSide chain forms disulfide bond;

Nle is L-nor-leucine;

D-hC is D-homocysteine

HC is L-homocysteine;

Nal is L-naphthylalanine;

Aib is 2-aminoisobutyric acid;

PE is L-Glutimic acid;

2. there is the polypeptide according to claim 1 of formula II:

Wherein X1 is the N-end of this polypeptide, and does not have or be selected from Q, A and pE;

X6 is S, s or a;

X7 is H, Aib or a;

X11 is D-Nle, Nle, M or f;

3. there is the polypeptide of the claim 1 of formula III:

X6 is S, s or a;

X7 is H, Aib or a;

X11 is D-Nle, Nle, M or f; And

X12 is selected from C, c, hC, D-hC; Wherein the side chain of the side chain of C, c, hC or D-hc and the C of X9, c, hC or D-hC forms two sulphurKey;

4. there is the polypeptide of the claim 1 of formula IV:

X6 is S, s or a;

X11 is D-Nle, Nle, M or f; And

X12 is selected from C, c, hC, D-hC; Wherein the side chain of the side chain of C, c, hC or D-hc and the C of X7, c, hC or D-hC forms two sulphurKey;

5. according to the polypeptide described in any one in claim 1,2,3 and 4, wherein X1 is pE; Or the acid amides of this polypeptide, ester orSalt.

6. according to the polypeptide described in any one in claim 1 to 4, wherein X1 does not exist; Or the acid amides of this polypeptide, ester orSalt.

7. according to the polypeptide described in any one in claim 1 to 4 and 6, wherein N-end is acid amides; Or the salt of this polypeptide.

8. according to the polypeptide described in embodiment 7, the wherein acid amides of N-end Shi Shi – NHR, and R be acetyl group, benzoyl,Phenacyl, succinyl group, caprylyl, 4-phenyl bytyry, 4-Cl-Ph-(CH₂)₃C (O)-or Ph-CH₂CH₂NHC(O)-；Or the salt of this polypeptide.

9. according to the polypeptide described in any one in claim 1 to 8, wherein X13 is F or f; Or the acid amides of this polypeptide, ester orSalt.

10. according to the polypeptide described in any one in claim 1 to 8, wherein X13 does not exist; Or the acid amides of this polypeptide, ester orSalt.

11. according to the polypeptide described in any one in claim 1 to 10, wherein C-end is acid amides; Or the salt of this polypeptide.

12. polypeptide according to claim 11, wherein acid amides and the R2 of C-end Shi Shi – C (O)-R2 are-NH₂、-NH-Me ,-NH-NHBn or-NH-(CH₂)₂-Ph; Or the salt of this polypeptide.

13. according to the polypeptide described in any one in claim 1-12, and wherein X6 is S; Or the acid amides of this polypeptide, ester or salt.

14. according to the polypeptide described in any one in claim 1-13, and wherein X7 is H; Or the acid amides of this polypeptide, ester or salt.

15. according to the polypeptide described in any one in claim 1 to 14, and wherein X11 is Nle or D-Nle; Or the acyl of this polypeptideAmine, ester or salt.

16. polypeptide according to claim 1, it is selected from:

Wherein use two amino acid represents of " * " mark to form the amino acid of disulphide; Or the acid amides of this polypeptide, ester or salt.

17. bioconjugates or its polymer, it comprises:

A. according to peptide or polypeptide or its acid amides, ester or the salt described in any one in claim 1 to 16, and

B. the part of prolong half-life;

The part of wherein said peptide or polypeptide and prolong half-life is or fusion covalently bound by connector optionally.

18. bioconjugates according to claim 17 or its polymer, wherein the part of prolong half-life is that IgG is constantTerritory or its fragment or human serum albumins.

19. according to the bioconjugates described in claim 17 or 18, and wherein the part of prolong half-life is to have LALA sudden changeThe Fc fragment that the FcLALA of (L234A, L235A) modifies.

20. bioconjugates according to claim 19, wherein the part of prolong half-life is Fc domain, and it is by connectingThe polypeptide of junctor and formula I, II, III or IV merges, and wherein connector has following formula:

-[GGGGS] n-, n is 2 or 3, and the polypeptide of formula I, II, III or IV contains naturally occurring amino acid.

21. bioconjugates according to claim 20, wherein polypeptide is the polypeptide that is selected from following formula I: QRPRC*SHKGPMC*F, QRPRLSHKC*PMC*F and QRPRLSC*KGPMC*F, wherein use two amino acid of " * " mark to represent to pass throughTheir side chain forms the amino acid of disulfide bond or amido link.

22. according to the bioconjugates described in claim 17 or 18 or its polymer, wherein the part of prolong half-life is peopleSeralbumin.

23. bioconjugates according to claim 22, wherein human serum albumins connects by the connector chemistry of following formulaBe connected to the N-end of the polypeptide of any same form in formula I to IV:

Wherein x is 1-20, and R is alkylidene, cycloalkyl, aryl or heteroaryl linear or side chain or its combination, and R ' is linearOr alkylidene, aryl or cycloalkyl or its combination of side chain.

24. according to the bioconjugates described in claim 17 or 18, wherein human serum albumins is by the connector of following formulaBe connected to the C-end of the polypeptide of any same form in formula I to IV:

25. treat or the disease of the excitement response of prevention to apj receptor or the method for illness in the individuality that has needs, and it comprisesTo described individual administering therapeutic effective dose according to the polypeptide described in any one or its acid amides in claim 1 to 24, ester orSalt or its bioconjugates.

26. the method for claim 25, wherein said disease or illness are selected from: acute decompensation DHF (ADHF),Chronic heart failure, pulmonary hypertension, auricular fibrillation, Bu Lugeda Cotard, Ventricular Tachycardia, atherosclerotic,Hypertension, ISR, ischemic angiocardiopathy, cardiomyopathy, cardiac fibrosis, cardiac arrhythmia, water retention, diabetes(comprising gestational diabetes mellitus), obesity, peripheral arterial disease, cerebrovas-cularaccident, temporary ischemic episode, traumatic brain injury, fleshAmyotrophic lateral sclerosis, burn (comprising sunburn) and pre-eclampsia.

27. according to polypeptide or its acid amides, ester or salt or its bioconjugates described in any one in claim 1 to 24, itsAs medicine.

28. according to polypeptide or its acid amides, ester or salt or its bioconjugates described in any one in claim 1 to 24, itsBe used for the treatment of or prevent disease or the illness of the excitement response to apj receptor.

29. according to polypeptide or its acid amides, ester or salt or its bioconjugates described in any one in claim 1 to 24, itsBe used for the treatment of acute decompensation DHF (ADHF), chronic heart failure, pulmonary hypertension, auricular fibrillation, Bu LugeReach Cotard, Ventricular Tachycardia, atherosclerotic, hypertension, ISR, ischemic angiocardiopathy, cardiac muscleDisease, cardiac fibrosis, cardiac arrhythmia, water retention, diabetes (comprising gestational diabetes mellitus), obesity, peripheral arterial disease, the cerebrovascularAccident, temporary ischemic episode, traumatic brain injury, ALS, burn (comprising sunburn) and pre-eclampsia.

30. combination products, its comprise treatment effective dose according to polypeptide or its acyl described in any one in claim 1 to 24The common activating agent of amine, ester or salt or its bioconjugates and one or more therapeutic activities.

31. according to the combination product of claim 30, wherein said activating agent is altogether selected from cardiotonic, Beta-3 adrenergic receptor resistanceDisconnected agent, HMG-Co-A reductase inhibitor, angiotensin II receptor antagonists, ACE (ACE) inhibitor,Calcium channel blocker (CCB), endothelin antagonist, renin inhibitor, diuretics, ApoA-I analogies, antidiabetic, fat-reducingAgent, aldosterone receptor blocking agent, blockade of endothelin receptors agent, aldosterone synthase inhibitors (ASI), CETP inhibitor, anti-coagulants,Relaxain, BNP (Nesiritide) and/or nep inhibitor.

32. pharmaceutical compositions, its comprise treatment effective dose according to the polypeptide described in any one in claim 1 to 24, itsAcid amides, ester or salt or its bioconjugates and one or more carriers of pharmaceutically accepting.